Amides of antibiotic GE 2270 factors

ABSTRACT

PCT No. PCT/EP92/00002 Sec. 371 Date Jul. 1, 1993 Sec. 102(e) Date Jul. 1, 1993 PCT Filed Jan. 2, 1992 PCT Pub. No. WO92/12172 PCT Pub. Date Jul. 23, 1992The present invention is directed to novel amide derivatives of antibiotic GE 2270 compounds and a process for preparing them. Said amide derivatives are antimicrobial agents active against gram positive bacteria as well as gram negative bacteria.

The present invention is directed to novel amide derivatives ofantibiotic GE 2270 having the following formula I ##STR1## wherein Rrepresents:

hydrogen, hydroxymethyl, or methoxymethyl;

R₁ represents:

hydrogen, or methyl:

Y represents:

a group of formula ##STR2## wherein: R₂ represents:

hydrogen, (C₁ -C₄)alkyl, amino(C₂ -C₄)alkyl, (C₁ -C₄)alkylamino-(C₁-C₄)alkyl, or di-(C₁ -C₄)alkylamino-(C₁ -C₄)alkyl;

R₃ represents:

hydrogen, a linear or branched (C₁ -C₁₄)alkyl group bearing from 1 to 3substituents selected from: carboxy, sulfo, phosphono, amino which maybe optionally protected with a lower alkoxycarbonyl or abenzyloxycarbonyl group, (C₁ -C₄)alkylamino wherein the alkyl moiety maybe optionally substituted with a carboxy group, di-(C₁ -C₄)alkylamino,hydroxy, halo, (C₁ -C₄)alkoxy wherein the alkyl moiety may be optionallysubstituted with a carboxy group, (C₁ -C₄)alkoxycarbonyl, mercapto, (C₁-C₄)alkylthio wherein the alkyl moiety may be optionally substitutedwith a carboxy group, phenyl which may be optionally substituted with 1to 3 substituents selected from carboxy, sulfo, hydroxy, halo andmercapto, carbamyl, (C₁ -C₆)alkylcarbamyl wherein the alkyl moiety maybe optionally substituted with 1 or 2 substituents selected fromcarboxy, amino, (C₁ -C₄)alkylamino and di-(C₁ -C₄)alkylamino, di-(C₁-C₄)alkylcarbamyl wherein the alkyl moieties together with the adjacentnitrogen atom may also represent a saturated 5-7 membered heterocyclicring which may optionally be substituted with a carboxy or a carbamylgroup on one of the ring carbons and may optionally contain a furtherheterogroup selected from O, S and N, benzoylamino wherein the phenylgroup may be substituted from 1 to 3 hydroxy group, a nitrogencontaining 5-6 membered heterocyclic ring which may be unsaturated,partially saturated or wholly saturated and may contain 1 to 3 furtherheteroatoms selected from N, S and O wherein one of the carbons of thering may optionally bear a group carboxy, sulfo, carboxy(C₁ -C₄)alkyland sulfo(C₁ -C₄)alkyl and the ring nitrogen atom may optionally besubstituted by (C₁ -C₄)alkyl, carboxy(C₁ -C₄)alkyl, sulfo(C₁ -C₄)alkyl,and benzyl;

(C₃ -C₆)alkenyl, optionally substituted by carboxy or sulfo;

1-deoxy-1-glucityl;

2-deoxy-2-glucosyl;

a fully saturated 5 to 7 membered nitrogen containing heterocyclic ringwherein the nitrogen atom may be optionally substituted by (C₁ -C₄)alkylor benzyl and one or two carbons of the ring skeleton may bear asubstituent selected from (C₁ -C₄)alkyl, carboxy and sulfo;

or

R₂ and R₃ taken together with the adjacent nitrogen atom represent afully saturated 5-7 membered heterocyclic ring which may optionallycontain a further heteroatom selected from O, S and N, and mayoptionally bear one or two substituents on the ring carbons selectedfrom (C₁ -C₄)alkyl, benzyl, carboxy, sulfo, carboxy(C₁ -C₄)alkyl, andsulfo(C₁ -C₄)alkyl;

R₄ represents

hydrogen, methyl, or hydroxymethyl

with the proviso that when R₄ is hydrogen or hydroxymethyl, thensimultaneously R is methoxymethyl and R₁ is methyl;

and the pharmaceutically addition salts thereof.

This invention includes also a process for preparing the compounds ofthis invention from the corresponding starting compounds of formula (II)##STR3## wherein W is a carboxylic function or an activated esterthereof.

Antibiotic GE 2270 is prepared by culturing a sample of Planobisporarosea ATCC 53773 or a producing variant or mutant thereof and isolatingthe desired antibiotic substance from the mycelium and/or thefermentation broth. Planobispora rosea ATCC 53773 was isolated from asoil sample and deposited on Jun. 14, 1988 with the American TypeCulture Collection (ATCC), 12301 Parklawn Drive, Rockville, Md. 20852Maryland, U.S.A., under the provisions of the Budapest Treaty.

The strain has been accorded accession number ATCC 53773.

Antibiotic GE 2270 factor A is the main component of the antibiotic GE2270 complex.

Antibiotic GE 2270 factor A and Planobispora rosea ATCC 53773 aredescribed in European Patent Application Publication No. 359062.

Recent studies showed that antibiotic GE 2270 factor A can berepresented by the following general formula III ##STR4##

When antibiotic GE 2270 factor A is treated under selective hydrolysisconditions some derivatives named antibiotic GE 2270 factor A₁, A₂ andA₃ are obtained. Said factors A₁, A₂ and A₃ and the hydrolysis processfor preparing then is disclosed in the European Patent ApplicationPublication No. 406745 and U.S. patent application Ser. No. 547,647,U.S. Pat. No. 5,139,778 which is hereby incorporated by reference.

Generally, the above mentioned hydrolytic conditions involve the use ofmixtures of buffered or unbuffered aqueous acid media and polar organicsolvents. The reaction temperature varies depending on factors such asthe strength and the concentration of the acid employed, and isgenerally comprised between -10° C. and 90° C. Also the reaction timevaries considerably depending on parameters such as the temperature, theacid strength and its concentration generally, it may vary from a fewminutes to several hours.

In general, when milder hydrolysis conditions are employed, e.g. shorterreaction time and lower temperature or lower acid strength orconcentration, antibiotic GE 2270 factor A₁ is normally obtained, whilestronger hydrolysis conditions yield antibiotic GE 2270 factor A₂. Toobtain antibiotic GE 2270 factor A₃, still more drastic hydrolysisconditions are necessary.

While antibiotic GE 2270 factors A₂ and A₃ can be directly utilized asthe starting materials for the production of the compounds of thisinvention, antibiotic GE 2270 factor A₁ is not suitable as the startingmaterial for direct production of the compounds of this invention;however, it can be utilized as a precursor of the said startingmaterials as it will be explained further.

Antibiotic GE 2270 factors A₂ and factor A₃ are characterized by havingan ester and a carboxy function respectively in the upper part of themolecule. In particular, it has been found that antibiotic GE 2270factor A₂ and factor A₃ can be represented by the above defined formulaII wherein:

W represents COOH (antibiotic GE 2270 factor A₃) or the ester moiety(antibiotic GE 2270 factor A₂) ##STR5## R is methoxymethyl, R₁ is methyland

R₄ is methyl.

Both antibiotic GE 2270 factor A₂ and factor A₃ (and mixture thereof)can be used as suitable starting materials for the production of thecompounds of the invention, even if factor A₃ is the preferred one.Factor A₂ may be employed directly as an activated ester or may beconverted to factor A₃ by drastic acid hydrolysis conditions, asmentioned above, or by basic hydrolysis with diluted alkali (asdescribed in European Patent Application Publication No. 406745 and U.S.patent application Ser. No. 547,647).

It was recently found (European Patent Application Publication No.451486and U.S. patent application Ser. No. 665,612) which is herebyincorporated by reference that other minor components can be isolatedfrom the cultures of Planobispora rosea ATCC 53773 or an antibiotic GE2270 producing variant or mutant thereof. In particular, they are foundin the mycelium and also in the fermentation broths of the culturedmicroorganism.

A preferred procedure for recovering said minor components of antibioticGE 2270 from the mycelium includes extracting the filtered orcentrifugated mycelium with a water-miscible organic solvent,concentrating the extracts and recovering the crude antibiotic substanceby precipitation, optionally with the addition of a precipitating agent,by extraction of the aqueous residue with a water-immiscible organicsolvent or by adsorption chromatography followed by elution of thedesired product from the absorption matrix.

It was recently found (European Patent Application No. 91114667.8),which correspond to U.S. patent application Ser. No. 07/931,084, filedAug. 17, 1992 which is hereby incorporated by reference that a furtherminor component (factor C_(2a)) can be isolated from the same culture ofPlanobispora rosea ATCC 53773 described above.

The physico-chemical characteristics of antibiotic GE 2270 C_(2a) arethe following:

A) The ultraviolet absorption spectrum recorded with a Perkin ElmerModel 320 spectrometer exhibit the following absorption maxima:

    ______________________________________                                        Solvent             UV max (nm)                                               ______________________________________                                        0.1M HCl            245-250 (shoulder)                                                            300-315                                                   0.1M KOH            245-250 (shoulder)                                                            300-315                                                   Phosphate buffer pH 7.38                                                                          245-250 (shoulder)                                                            300-315                                                   Methanol            245-250 (shoulder)                                                            300-315                                                   ______________________________________                                    

B) The ¹ H-NMR spectrum of antibiotic GE 2270 factor C_(2a) was recordedat 250 MHz with a Bruker spectrometer. The spectrum of the antibioticDNSO-d₆ (hexadeuterodimethylsulfoxide) using TMS as the internalstandard (0.00 ppm) exhibits following groups of signals [δ, ppm, m](s=singlet, d=doublet, t=triplet, m=multiplet, Py=pyridine, Tz=thiazole)

9.03, d, (NH); 8.70, d, (2NH's); 8.60, s, 8.54, s, 8.29, s, and 7.38, s,(Tz CH's): 8.48, m, (glycine NH); 8.43, d, and, 8.27, d, (Py CH's);7.35-7.20, m, (aromatic CH's and primary amide NH); 6.98, s (primaryamide NH); 6.04, d, (OH); 5.80, t (OH); 5.35-5.15, m, (αCH's); 5.04, m,(phenylserine βCH); 4.98, s [CH₂ (OCH₃)]; 4.87, d, [CH₂ (OH)]; 4.81, mand 4.56, m, (oxazoline CH₂); 4.35-3.75, m, (CH₂ of glycine andprolineamide CH's); 3.39,s, (OCH₃); 2.71, m, and 1.30, m, (CH₂ ofasparagine); 2.48, d, (NCH₃ of N-methylasparagine); 2.22-1.80, m,(isopropyl CH and prolineamide CH's); 0.88 and 0.84, d, (valine CH₃ 's)

C) Antibiotic GE 2270 factor C_(2a) shows retention time (R_(t)) of 12.6min and retention time relative to antibiotic GE 2270 factor A (R_(t)16.6 min) of 0.76 when analyzed with the following reverse phase HPLCsystem:

Column: Bakerbond® C8 (5 μm) 4.6×250 mm (Bakerbond® is a trade name forreverse phase octylsilyl silica gel HPLC columns supplied by J. T. BakerResearch Product, Phillisburg, N.J. 08865 USA)

Flow rate: 1.8 ml/min

Phase A: CH₃ CN:tetrahydrofuran:40 mM HCOONH₄ 40:40:20

Phase B: CH₃ CN:tetrahydrofuran:40 mM HCOONH₄ 10:10:80

Elution: linear gradient from 20% to 30% of Phase A in 20 min

Detection: UV 254 nm

D) The main FAB-MS peak of antibiotic GE 2270 factor C_(2a) is 1306daltons. This corresponds most likely to the lowest isotope of theprotonated molecular ion. The analysis was performed on a Kratos MS-50double focusing mass spectrometer, using 8 kV accelerating voltage and asaddle field atom gun with Xe gas (2×10⁻⁵ torr pressure indicated on thesource ion gauge) at 6 kV voltage and 1 mA current. The antibiotic forthe FAB-MS analysis was mixed with a thioglycerol matrix containing 0.1Macetic acid.

Some of said minor components of antibiotic GE 2270 (i.e. factors B₁,B₂, C₁, C₂, C_(2a), D₁ D₂ and E) may be represented by the generalformula II mentioned above wherein

W represents the moiety: ##STR6## R represents respectively hydrogen forGE 2270 factors C₁ and D₁ methyl for factor B₂, hydroxymethyl forfactors D₂ and E and methoxymethyl for factors B₁, C₂ and C_(2a) ;

R₁ represents hydrogen for GE 2270 factors B₁, D₁ and E and methyl forGE 2270 factors B₂, C₁, C_(2a) and D₂ ; and

R₄ represents hydrogen for GE 2270 factor C₂, methyl for GE 2270 factorsB₁, B₂, C₁, D₁, D₂ and E and hydroxymethyl for factor C_(2a).

When antibiotic GE 2270 factors D₁, D₂ and E or mixture thereof aretreated by the same hydrolytic process outlined above (and described inEuropean Patent Application Publication No. 406745 and U.S. patentapplication Ser. No. 547,647) for preparing antibiotic GE 2270 factorsA₂ and A₃ from antibiotic GE 2270 factor A, the common moiety W citedabove is hydrolyzed to a carboxy moiety leaving the substituents R, R₁and R₄ unaltered.

Therefore, the derivatives of formula II wherein W is a carboxy or anactivated ester function, R is hydrogen, hydroxymethyl or methoxyethyl,R₁ is hydrogen or methyl and R₄ is hydrogen, methyl or hydroxymethyl,with the proviso that when R₄ is hydrogen or hydroxymethyl then R ismethoxymethyl and R₁ is methyl, can be used as starting material of thepresent invention. It has to be clear that as with other microorganisms,the characteristics of the GE 2270 producing strains are subject tovariation. For example, artificial variants and mutants of the straincan be obtained by treatment with various known mutagens, such as U.V.rays, X-rays, high frequency waves, radioactive rays, and chemicals suchas nitrous acid, N-methyl-N'-nitro-N-nitroso-guanidine, and many others.All natural and artificial variants and mutants which belong to aspecies of the genus Planobispora and produce antibiotic GE 2270 aredeemed equivalent to strain Planobispora rosea ATCC 53773 for thepurposes of this invention.

As used herein, the term "alkyl", either alone or in combination withother substituents, includes both straight and branched hydrocarbonsgroups; more particularly, "(C₁ -C₁₄)alkyl" represents a straight orbranched aliphatic hydrocarbon chain of 1 to 14 carbon atoms such asmethyl, ethyl, propyl, 1-methylethyl, butyl, 1-methylpropyl,1,1-dimethylethyl, pentyl, 1-methylbutyl, 2-methylbutyl, 1-hexyl,2-hexyl, 3-hexyl, 3,3-dimethyl-1-butyl, 4-methyl-1-pentyl and3-methyl1-pentyl, heptyl, octyl, nonyl, decyl, undecyl, dodecyl,tridecyl and tetradecyl; likewise, "(C₁ -C₄)alkyl" represents a straightor branched hydrocarbon chain of 1 to 4 carbon atoms such as those alkylof 1 to 4 carbons exemplified above.

As described above the "(C₁ -C₁₄)alkyl" moiety may bear 1 to 3substituents.

The term "halo" represents a halogen atom radical selected from fluoro,chloro, bromo and iodo.

As used herein, the term "(C₃ -C₆)alkenyl" means an alkylene radicalhaving three to six carbon atoms and a double bond; it comprisespropenyl, 3-butenyl, 2-butenyl, 2-methylpropenyl, 2-pentenyl, 3-hexenyland so on, which may be optionally substituted with a carboxy or a sulfogroup.

The expression "a nitrogen containing 5-6 membered heterocyclic ringwhich may contain 1 to 3 further heteroatoms selected from N, S and O"according to the present invention includes unsaturated, partiallysaturated and wholly saturated ring systems such as pyridine,pyrimidine, pyrazine, pyrrolidine, piperidine, piperazine, oxazole,oxazoline, oxazolidine, pyrazoline, pyrazolidine, thiazolidine,morpholine, thiomorpholine, pyrrole, pyrroline, imidazole,imidazolidine, thiadiazole, oxadiazole and tetrazole.

In said "nitrogen containing 5-6 membered heterocyclic ring" 1 to 3 ringcarbons may optionally bear a group carboxy, sulfo, carboxy(C₁ -C₄)alkyland sulfo(C₁ -C₄)alkyl and the ring nitrogen atom may optionally besubstituted by (C₁ -C₄)alkyl, carboxy(C₁ -C₄)alkyl, sulfo(C₁ -C₄)alkyl,and benzyl,

The expression "fully saturated 5-7 membered nitrogen containingheterocyclic ring wherein the nitrogen atom may be optionallysubstituted by (C₁ -C₄)alkyl or benzyl" identifies a fully saturatedheterocycle of 5-7 members containing a nitrogen atom which can beoptionally substituted by (C₁ -C₄)alkyl or benzyl wherein the carbonskeleton may optionally bear one or two substituents selected from (C₁-C₄)alkyl, carboxy and sulfo. Said heterocyclic rings are connected withthe nitrogen moiety of the rest ##STR7## through a bond between the samenitrogen moiety and a carbon atom of the heterocyclic rest. Examples ofsaid radicals are: 1-methyl-4-pyrrolidinyl, 3-piperidinyl,1-ethyl-4-piperidinyl, 1-benzyl-2,6-dimethyl-4-piperidinyl, and4-carboxy-1-methyl-2-piperidinyl;

When R₂ and R₃ taken together with the adjacent nitrogen atom represent"a fully saturated 5-7 membered heterocyclic ring which may optionallycontain a further heteroatom selected from O, S and N" this expressionincludes, for instance, the following heterocyclic groups: pyrrolidino,morpholino, piperidino, piperazino, thiomorpholino, pyrazolidino,1,3-oxazolidino, 1,3-thiazolidino and hexahydroazepino When the furtherheteroatom is N it may optionally bear a substituent selected from (C₁-C₄)alkyl, benzyl, carboxy, carboxy(C₁ -C₄)alkyl, sulfo and sulfo(C₁-C₄)alkyl.

The term "1-deoxy-1-glucityl" identifies a compound of formula (I)wherein Y is a radical deriving from glucamine, i.e.1-amino-1-deoxy-glucitol. The term "2-deoxy-2-glucosyl" identifies acompound of formula (I) wherein Y is a radical deriving fromglucosamine, i.e. 2-amino-2-deoxyglucose.

A preferred group of compounds of the invention is represented by thosecompounds of formula I wherein R represents methoxymethyl, R₁ and R₄represent a methyl group and the other substituents are as definedabove.

A further preferred group of compounds of the invention are thosecompounds of formula I wherein R represents methoxymethyl, R₁ and R₄represent a methyl group, and Y represents a group of formula ##STR8##wherein R₂ is hydrogen and R₃ is defined as above.

A further preferred group of compounds of the invention is representedby those compounds of formula I wherein R is methoxymethyl, R₁ and R₄represent a methyl group and Y is an amino moiety which derive from anatural amino acid such as for example glycine, ornithine, serine,aspartic acid, tyrosine, leucine, phenylalanine, methionine, proline,threonine, lysine, or a synthetic dipeptide such as glycyllysine,serylproline, glycylprolinamide, tyrosylprolinamide,threonylprolinamide, leucylprolinamide.

A further preferred group of compounds comprises those compounds offormula I wherein R is methoxymethyl, R₁ and R₄ are methyl, Y is a groupNR₂ R₃ wherein R₂ is hydrogen and R₃ is a linear alkyl chain preferablyof 3 to 12 carbons, more preferably of 3 to 7 carbons substituted with agroup selected from COOH, SO₃ H and PO₃ H₂.

The most preferred compound is represented the formula I wherein R ismethoxymethyl, R₁ and R₄ are methyl and Y is a group NR₂ R₃ wherein R₂is hydrogen and R₃ is CH₂ CH₂ CH₂ CH₂ CH₂ --COOH.

A further preferred group of compounds of the invention are thosecompounds of formula I wherein R represents hydrogen, hydroxymethyl andmethoxymethyl, R₁ represents hydrogen or a methyl group, and Yrepresents a group of formula ##STR9## wherein R₂ is hydrogen and R₃ andR₄ are defined as above.

A further preferred group of compounds of the invention is representedby those compounds of formula I wherein R is hydrogen, hydroxymethyl ormethoxymethyl, R₁ represents hydrogen or a methyl group, R₄ is hydrogen,methyl or hydroxymethyl with the proviso that when R₄ is hydrogen orhydroxymethyl then R is methoxymethyl and R₁ is methyl, and Y is anamino moiety which derive from a natural amino acid such as for exampleglycine, ornithine, serine, aspartic acid, tyrosine, leucine,phenylalanine, methionine, proline, threonine, lysine, or a syntheticdipeptide such as glycyllysine, serylproline, glycylprolinamide,tyrosylprolinamide, threonylprolinamide, leucylprolinamide.

A further preferred group of compounds comprises those compounds offormula I wherein R is hydrogen, hydroxymethyl or methoxymethyl, R₁ ishydrogen or methyl, R₄ is hydrogen, methyl or hydroxymethyl with theproviso that when R₄ is hydrogen or hydroxymethyl then R ismethoxymethyl and R₁ is methyl, Y is a group NR₂ R₃ wherein R₂ ishydrogen and R₃ is a linear alkyl chain preferably of 3 to 12 carbons,more preferably of 3 to 7 carbons substituted with a group selected fromCOOH, SO₃ H and PO₃ H₂.

The last preferred group of compounds is represented by the formula Iwherein R hydrogen, hydroxymethyl or methoxymethyl, R₁ is hydrogen ormethyl, R₄ is as defined above and Y is a group NR₂ R₃ wherein R₂ ishydrogen and R₃ is CH₂ CH₂ CH₂ CH₂ CH₂ --COOH.

Representative examples of the compounds of the invention, include thosecompounds of formula I wherein R, R₁, R₄ and Y are as defined above and##STR10## represents ##STR11##

The compounds of the invention can form salts according to conventionalprocedures.

In particular, those compounds of formula I wherein the group --NR₂ R₃contains further amine functions form acid addition salts.

In addition, those compounds of the invention which contain acidfunctions in the --NR₂ R₃ moiety may also form base addition salts.

In general, those compounds of the invention which contain acid andbasic functions can form internal salts. For the scope of the presentinvention the "internal salts" are encompassed by the definition the"non-salt" form.

Preferred addition salts of the compounds of this invention are thepharmaceutically acceptable acid and/or base addition salts.

With the term "pharmaceutically acceptable acid and/or base additionsalts" are intended those salts with acids and/or bases which frombiological, manufacturing and formulation standpoint are compatible withthe pharmaceutical practice as well as with the use in the animal growthpromotion.

Representative and suitable acid addition salts of the compounds offormula I include those salts formed by standard reaction with bothorganic and inorganic acids such as, for example, hydrochloric,hydrobromic, sulfuric, phosphoric, acetic, trifluoroacetic,trichloroacetic, succinic, citric, ascorbic, lactic, maleic, fumaric,palmitic, cholic, pamoic, mucic, glutamic, camphoric, glutaric,glycolic, phthalic, tartaric, lauric, stearic, salicylic,methanesulfonic, dodecylsulfonic acid (estolic acid), benzenesulfonic,sorbic, picric, benzoic, cinnamic and the like acids.

Representative examples of these bases are: alkali metal oralkaline-earth metal hydroxide such sodium, potassium, and calciumhydroxide; ammonia and organic aliphatic, alicyclic or aromatic aminessuch as methylamine, dimethylamine, trimethylamine,2-amino-2-hydroxymethyl-1,3-propanediol (TRlS), picoline and basicaminoacids such as lysine, ornithine, arginine and histidine.

The transformation of the free amino or non-salt compounds of theinvention into the corresponding addition salts, and the reverse, i.e.the transformation of an addition salt of a compound of the inventioninfo the non-salt or free amino form, are within the ordinary technicalskill and are encompassed by the present invention.

For instance, a compound of formula I can be transformed into thecorresponding acid or base addition-salt by dissolving the non-salt formin an aqueous solvent and adding a slight molar excess of the selectedacid or base. The resulting solution or suspension is then lyophilizedto recover the desired salt. Instead of lyophilizing, in some instances,it is possible to recover the final salt by extraction with an organicsolvent, concentration to a small volume of the separated organic phaseand precipitation by adding a non-solvent.

In case the final salt is unsoluble in an organic solvent where thenon-salt form is soluble it is recovered by filtration from the organicsolution non-salt form after addition of the stoichiometric amount or aslight molar excess of the selected acid base.

The non-salt form can be prepared from a corresponding acid or base saltdissolved in an aqueous solvent which is then neutralized to free thenon-salt form. This is then recovered for instance by extraction with anorganic solvent or is transformed into another base or acid additionsalt by adding the selected acid or base and working up as above.

When following the neutralization desalting is necessary, a commondesalting procedure may be employed.

For example, column chromatography on controlled pore polydextraneresins (such as Sephadex LH 20) or silanized silica gel may beconveniently used. After eluting the undesired salts with an aqueoussolution, the desired product is eluted by means of linear gradient orstep-gradient of a mixture of water and a polar or apolar organicsolvent, such as acetonitrile/water from 50:50 to about 1001%acetonitrile.

As is known in the art, the salt formation either with pharmaceuticallyacceptable acids (bases) or non-pharmaceutically acceptable acids(bases) may be used as a convenient purification technique. Afterformation and isolation, salt form of a compound of formula I can betransformed into the corresponding non-salt or into a pharmaceuticallyacceptable salt.

In some instances the acid addition salt of a compound of formula I ismore soluble in water and hydrophilic solvents and has an increasedchemical stability.

However, in view of the similarity of the properties of the compounds offormula I and their salts, what is said in the present application whendealing with the biological activities of the compounds of formula Iapplies also to their pharmaceutically acceptable salts, and viceversa.

In view of their properties, the compounds of the invention can be usedas active ingredients in the preparation of medicaments for human oranimal treatment.

In particular, the amide derivatives of the antibiotic GE 2270 compoundsof formula I are antimicrobial agents mainly active against grampositive bacteria and gram positive as well as gram negative anaerobes.

A general procedure for preparing a compound of this invention isrepresented by the reaction (amidation) of a suitable antibiotic GE 2270compound having formula (II) ##STR12## wherein W represents a carboxy oran activated ester function;

R represents hydrogen, hydroxymethyl or methoxymethyl;

R₁ represents hydrogen or methyl;

R₄ represents hydrogen, methyl or hydroxymethyl,

with the proviso that, when R₄ represents hydrogen or hydroxymethyl,then simultaneously R is methoxymethyl and R₁ is methyl; with a selectedamine of formula HNR₂ R₃ wherein R₂ and R₃ have the same meanings asabove in an inert organic solvent and, when W is carboxy, in thepresence of a condensing agent.

In carrying out the amidation for preparing the compounds of thisinvention, sometimes, it is convenient to protect the functions of thereactants which are not involved in the amidation reaction but couldresult sensitive to the reaction conditions or negatively affect thereaction course, for instance, yielding undesired side-product.

Furthermore, when the amino acid contains further reactive functionssuch as amino, carboxy or mercapto groups which may interfere with thecourse of the amidation, these are protected by means of methods knownper se in the art such as those described in references books like E.Gross and J. Meienhofer "The Peptides", Vol. 3, Academic Press, NewYork, 1981 and M. Bodanszky and A. Bodanszky "The Practice of PeptideSynthesis, Springer-Verlag, Berlin Heidelberg, 1984. These protectinggroups must be stable at the conditions the amidation reaction takesplace and must be easily removable at the end of the reaction withoutaffecting either the newly formed amide bond or any other part of themolecule.

Representative examples of N-protecting groups which may beadvantageously used in the process of the invention for protecting anamino function are carbamate forming reagents characterized by thefollowing oxycarbonyl groups: 1,1-dimethylpropynyl-oxycarbonyl,t-butyloxycarbonyl, vinyloxycarbonyl, aryloxycarbonyl,cinnamyloxycarbonyl, benzyloxycarbonyl,p-nitrobenzyloxycarbonyl-3,4-dimethoxy-6-nitrobenzyloxycarbonyl,2,4-dichlorobenzyloxycarbonyl, 5-benzisoxazolylmethyloxycarbonyl,9-anthranylmethyloxycarbonyl, diphenylmethyloxycarbonyl,isonicotinyloxycarbonyl, diphenylmethyloxycarbonyl,isonicotinyloxycarbonyl, S-benzyloxycarbonyl, and the like.

A suitable protection for reactive carboxylic acid function is, forinstance, by forming an ester function.

The man skilled in the art is capable, also on the basis of the presentdisclosure, of deciding which functions of the amine HNR₂ R₃ need to beprotected, how they must be protected and the proper deprotectionreaction which is necessary to free the final compound.

As it is appreciated by the skilled technician, the ultimate choice ofthe specific protecting group depends on the characteristics of theparticular amide derivative which is desired. In fact, this amidefunction of the final compound should be stable at the condition ofremoval of the protecting group(s).

Since the conditions of removal of the different protecting groups areknown, the skilled technician capable of selecting the proper protectinggroup.

Inert organic solvents useful for the condensation reaction are thosesolvents which do not unfavorably interfere with the reaction course andare capable of at least partially solubilizing the antibiotic startingmaterial.

Examples of said inert solvents are organic amides, ethers of glycolsand polyols, phosphoramides, sulfoxides. Preferred examples of inertsolvents are: dimethylformamide, dimethoxyethane,hexamethylphosphoramide, dimethylsulfoxide, dioxane, and mixturesthereof.

Sometimes, water is compatible with the reaction conditions.

The condensing agent in the process of the invention when W is carboxyis one suitable for forming amide bonds in organic compounds and inparticular peptide synthesis.

Representative and preferred examples of condensing agents are (C₁-C₄)alkyl, phenyl or heterocyclic phosphorazidates such as,diphenylphosphorazidate (DPPA), diethyl-phosphorazidate,di(4-nitrophenyl)phosphorazidate, dimorpholylphosphorazidate anddiphenylphosphorochloridate orbenzotriazol-1-yl-oxy-tripyrrolidinophosphoniumhexafluorophosphate(PyBOP). The preferred condensing agent is diphenyl phosphorazidate(DPPA).

In the process of the invention, the amine reactant HNR₂ R₃ is normallyused in a slight molar excess.

In general, a 1- to 2-fold molar excess is used while a 1.2- to 1.5-foldmolar excess is preferred.

For the amidation to proceed, it is necessary that the amine HNR₂ R₃ becapable of forming a salt wish the carboxy function of the antibioticstarting material. In case the amine HNR₂ R₃ is not strong enough toform such a salt in the selected reaction medium, it is necessary to adda salt-forming base to the reaction mixture at least in an equimolecularamount with the antibiotic starting material.

Examples of said salt-forming bases are tertiary organic aliphatic oralicyclic amines such as trimethylamine, triethylamine, N-methylpyrrolidine or heterocyclic bases such as picoline, and the like.

The condensing agent is generally employed in a slight molar excess suchas from 1.1 to 1.5 and preferably is 1.2 times the antibiotic GE 2270starting compound.

In addition, the amine reactant HNR₂ R₃ may also conveniently beintroduced in the reaction medium as corresponding acid addition salt,e,g. the hydrochloride. In this case, at least a double molar proportionand preferably a 2 to 3 fold molar excess of a strong base capable offreeing the HNR₂ R₃ amine from its salts, is used. Also in this case,the suitable base is a tertiary organic aliphatic or alicyclic aminelike those exemplified above. In fact, at least in some instances, theuse of salt of the amine HNR₂ R₃, which then freed in situ with theabove mentioned bases, is greatly preferred especially when the salt ismore stable than the corresponding free amine.

The reaction temperature will vary considerably depending on thespecific starting materials and reaction conditions. In general, it ispreferred to conduct the reaction at temperatures between 0°-20° C.

Also the reaction time vary considerably depending on the other reactionparameters. In general the condensation reaction is completed in about5-24 h.

In any case, the reaction course is monitored by TLC or preferably byHPLC according to methods known in the art.

On the basis of the results of these assays a man skilled in the artwill be able to evaluate the reaction course and decide when to stop thereaction and start working up the reaction mass according to known perse techniques which include, for instance, extraction with solvents,precipitation by addition non-solvents, etc., in conjunction withfurther separations and purifications by column chromatography.

As already said, when protection of the HNR₂ R₃ reactant is necessary,the protected final compound then de-protected according to procedureswhich are known per se and mainly depends on the protecting groupinvolved.

When an activated ester is used as the GE 2270 starting material, saidester is one wherein the esterified alcohol is providing a leaving groupwhich can be readily displaced and substituted by the amine HNR₂ R₃under reaction conditions which do not modify the other portions of themolecule. The amine reactant is usually employed in a molar excess overthe activated ester in a solvent which is selected from those mentionedabove and the lower alkanols. The reaction temperature generally rangesbetween 0° C. and 100° C. Examples of the activated ester include loweralkyl esters wherein the lower alkyl moiety is optionally substituted bycyano and nitro, phenyl esters substituted by halo and nitro groups aswell as the ester moiety contained in GE 2270 factor A₂.

It is evident that in many instances a compound of the invention may beprepared in more than one way and that a compound of the invention maybe transformed into another by means of known per se reactions.

For instance when the HNR₂ R₃ amine contains a carboxy or an esterfunction which can be further converted into the corresponding amidederivative, a desired compound of formula I may be prepared bycondensing first said amine with the selected GE 2270 starting materialand then converting the carboxy or ester function to amide by reactionwith the appropriate amine.

The following tables list the structure formulas of some representativecompounds of the invention (TABLE I) and their methods of preparation,(described in details in the Experimental Section), starting materialsand reaction yields (TABLE II).

                                      TABLE I                                     __________________________________________________________________________    COMPOUND OF                                                                   EXAMPLE NO.                                                                             Y                           R      R.sub.1                                                                          R.sub.4                       __________________________________________________________________________     1        NHCH.sub.2 COOH             CH.sub.2 OCH.sub.3                                                                   CH.sub.3                                                                         CH.sub.3                       2                                                                                       ##STR13##                  CH.sub.2 OCH.sub.3                                                                   CH.sub.3                                                                         CH.sub.3                       3                                                                                       ##STR14##                  CH.sub.2 OCH.sub.3                                                                   CH.sub.3                                                                         CH.sub.3                       4                                                                                       ##STR15##                  CH.sub.2 OCH.sub.3                                                                   CH.sub.3                                                                         CH.sub.3                       5                                                                                       ##STR16##                  CH.sub.2 OCH.sub.3                                                                   CH.sub.3                                                                         CH.sub.3                       6                                                                                       ##STR17##                  CH.sub.2 OCH.sub.3                                                                   CH.sub.3                                                                         CH.sub.3                       7                                                                                       ##STR18##                  CH.sub.2 OCH.sub.3                                                                   CH.sub.3                                                                         CH.sub.3                       8                                                                                       ##STR19##                  CH.sub.2 OCH.sub.3                                                                   CH.sub.3                                                                         CH.sub.3                       9                                                                                       ##STR20##                  CH.sub.2 OCH.sub.3                                                                   CH.sub.3                                                                         CH.sub.3                      10                                                                                       ##STR21##                  CH.sub.2 OCH.sub.3                                                                   CH.sub.3                                                                         CH.sub.3                      11                                                                                       ##STR22##                  CH.sub.2 OCH.sub.3                                                                   CH.sub.3                                                                         CH.sub.3                      12                                                                                       ##STR23##                  CH.sub.2 OCH.sub.3                                                                   CH.sub.3                                                                         CH.sub.3                      13                                                                                       ##STR24##                  CH.sub.2 OCH.sub.3                                                                   CH.sub.3                                                                         CH.sub.3                      14                                                                                       ##STR25##                  CH.sub.2 OCH.sub.3                                                                   CH.sub.3                                                                         CH.sub.3                      15                                                                                       ##STR26##                  CH.sub.2 OCH.sub.3                                                                   CH.sub.3                                                                         CH.sub.3                      16                                                                                       ##STR27##                  CH.sub.2 OCH.sub.3                                                                   CH.sub.3                                                                         CH.sub.3                      17                                                                                       ##STR28##                  CH.sub.2 OCH.sub.3                                                                   CH.sub.3                                                                         CH.sub.3                      18        NHCH.sub.2 CH.sub.2 CH.sub.2 COOH                                                                         CH.sub.2 OCH.sub.3                                                                   CH.sub.3                                                                         CH.sub.3                      19        NHCH.sub.2 CH.sub.2 CH.sub.2 CH.sub.2 CH.sub.2 COOH                                                       CH.sub.2 OCH.sub.3                                                                   CH.sub.3                                                                         CH.sub.3                      20        NHCH.sub.2 CH.sub.2 CH.sub.2 CH.sub.2 CH.sub.2 CH.sub.2                       CH.sub.2 COOH               CH.sub.2 OCH.sub.3                                                                   CH.sub.3                                                                         CH.sub.3                      21        NHCH.sub.2 CH.sub.2 CH.sub.2 CH.sub.2 CH.sub.2 CH.sub.2                       CH.sub.2 CH.sub.2 CH.sub.2 CH.sub.2 COOH                                                                  CH.sub.2 OCH.sub.3                                                                   CH.sub.3                                                                         CH.sub.3                      22        NHCH.sub.2 CH.sub.2 SO.sub.3 H                                                                            CH.sub.2 OCH.sub.3                                                                   CH.sub.3                                                                         CH.sub.3                      23        NHCH.sub.2 CH.sub.2 CH.sub.2 SO.sub.3 H                                                                   CH.sub.2 OCH.sub.3                                                                   CH.sub.3                                                                         CH.sub.3                      24        NHCH.sub.2 CH.sub.2 CH.sub.2 PO.sub.3 H.sub.2                                                             CH.sub.2 OCH.sub.3                                                                   CH.sub.3                                                                         CH.sub.3                      25        NHCH.sub.2 CH.sub.2 CH.sub.2 CH.sub.2 CH.sub.2 PO.sub.3                                                   CH.sub.2 OCH.sub.3                                                                   CH.sub.3                                                                         CH.sub.3                      26                                                                                       ##STR29##                  CH.sub.2 OCH.sub.3                                                                   CH.sub.3                                                                         CH.sub.3                      27                                                                                       ##STR30##                  CH.sub.2 OCH.sub.3                                                                   CH.sub.3                                                                         CH.sub.3                      28                                                                                       ##STR31##                  CH.sub.2 OCH.sub.3                                                                   CH.sub.3                                                                         CH.sub.3                      29                                                                                       ##STR32##                  CH.sub.2 OCH.sub.3                                                                   CH.sub.3                                                                         CH.sub.3                      30                                                                                       ##STR33##                  CH.sub.2 OCH.sub.3                                                                   CH.sub.3                                                                         CH.sub.3                      31                                                                                       ##STR34##                  CH.sub.2 OCH.sub.3                                                                   CH.sub.3                                                                         CH.sub.3                      32        NHCH.sub.2 CH.sub.2 N(CH.sub.3).sub.2                                                                     CH.sub.2 OCH.sub.3                                                                   CH.sub.3                                                                         CH.sub.3                      33                                                                                       ##STR35##                  CH.sub.2 OCH.sub.3                                                                   CH.sub.3                                                                         CH.sub.3                      34                                                                                       ##STR36##                  CH.sub.2 OCH.sub.3                                                                   CH.sub.3                                                                         CH.sub.3                      35        NH.sub.2                    CH.sub.2 OCH.sub.3                                                                   CH.sub.3                                                                         CH.sub.3                      36        NHCH.sub.2 CH.sub.2 CH.sub.2 NH.sub.2                                                                     CH.sub.2 OCH.sub.3                                                                   CH.sub.3                                                                         CH.sub.3                      37        NHCH.sub.2 CHO              CH.sub.2 OCH.sub.3                                                                   CH.sub.3                                                                         CH.sub.3                      38        NHCH.sub.2 CH.sub.2 NHCH.sub.2 CH.sub.2 COOH                                                              CH.sub.2 OCH.sub.3                                                                   CH.sub.3                                                                         CH.sub.3                      39        NHCH.sub.2 CH.sub.2 SCH.sub.2 CH.sub.2 COOH                                                               CH.sub.2 OCH.sub.3                                                                   CH.sub.3                                                                         CH.sub.3                      40        NHCH.sub.2 CH.sub.2 CH.sub.2 CHCHCOOH                                                                     CH.sub.2 OCH.sub.3                                                                   CH.sub.3                                                                         CH.sub.3                      41        NHCH.sub.2 CH.sub.2 OCH.sub.2 CH.sub.2 COOH                                                               CH.sub.2 OCH.sub.3                                                                   CH.sub.3                                                                         CH.sub.3                      42        NHCH.sub.2 CH.sub.2 CH.sub.2 CH.sub.2 CH.sub.2 COOH                                                       CH.sub.2 OCH.sub.3                                                                   CH.sub.3                                                                         CH.sub.2 OH                   43        NHCH.sub.2 CH.sub.2 CH.sub.2 CH.sub.2 CH.sub.2 COOH                                                       H      H  CH.sub.3                      44        NHCH.sub.2 CH.sub.2 CH.sub.2 CH.sub.2 CH.sub.2 COOH                                                       CH.sub.2 OH                                                                          CH.sub.3                                                                         CH.sub.3                      __________________________________________________________________________     Compounds No. 2, 11, 12, 34, 36 were isolated as trifluoroacetate salts  

                                      TABLE II                                    __________________________________________________________________________    COMPOUND OF                                                                              STARTING MATERIALS                                                 EXAMPLE NO.                                                                              (GE 2270 FACTOR + AMINE REACTANT)                                                                              METHOD OVERALL                    __________________________________________________________________________                                                       YIELD                       1         A.sub.3 + HCl.NH.sub.2 CH.sub.2 COOEt                                                                          A.sub.1                                                                              80%                         2                                                                                        ##STR37##                       A.sub.1                                                                              72%                         3                                                                                        ##STR38##                       A.sub.1                                                                              70%                         4                                                                                        ##STR39##                       A.sub.1                                                                              54%                         5                                                                                        ##STR40##                       A.sub.1                                                                              70%                         6                                                                                        ##STR41##                       A.sub.1                                                                              70%                         7                                                                                        ##STR42##                       A.sub.1                                                                              60%                         8                                                                                        ##STR43##                       A.sub.1                                                                              70%                         9                                                                                        ##STR44##                       A.sub.1                                                                              75%                        10                                                                                        ##STR45##                       A.sub.1                                                                              70%                        11                                                                                        ##STR46##                       B.sub.1                                                                              64%                        12                                                                                        ##STR47##                       B.sub.1                                                                              74%                        13                                                                                        ##STR48##                       C.sub.1                                                                              70%                        14                                                                                        ##STR49##                       C      83%                        15                                                                                        ##STR50##                       A      60%                                    ##STR51##                       C      70%                        16                                                                                        ##STR52##                       C      60%                        17                                                                                        ##STR53##                       C      65%                        18         A.sub.3 + HCl.NH.sub.2 CH.sub.2 CH.sub.2 CH.sub.2 COOMe                                                        A.sub.1                                                                              73%                        19         A.sub.3 + HCl.NH.sub.2 CH.sub.2 CH.sub.2 CH.sub.2 CH.sub.2                    CH.sub.2 COOMe                   A.sub.1                                                                              77%                                   A.sub.3 + NH.sub.2 CH.sub.2 CH.sub.2 CH.sub.2 CH.sub.2                        CH.sub.2 COOH                    B      70%                        20         A.sub.3 + PTSA.NH.sub.2 CH.sub.2 CH.sub.2 CH.sub.2 CH.sub.2                   CH.sub.2 CH.sub.2 CH.sub.2 COOMe A.sub.1                                                                              75%                        21         A.sub.3 + PTSA.NH.sub.2 CH.sub.2 CH.sub.2 CH.sub.2 CH.sub.2                   CH.sub.2 CH.sub.2 CH.sub.2 CH.sub.2 CH.sub.2 CH.sub.2 COOMe                                                    A.sub.1                                                                              70%                        22         A.sub.3 + NH.sub.2 CH.sub.2 CH.sub.2 SO.sub.3 H                                                                B      20%                        23         A.sub.3 + NH.sub.2 CH.sub.2 CH.sub.2 CH.sub.2 SO.sub.3                                                         B      25%                        24         A.sub.3 + NH.sub.2 CH.sub.2 CH.sub.2 CH.sub.2 PO.sub.3                                                         B.sub.2                                                                              40%                        25         A.sub.3 + NH.sub.2 CH.sub.2 CH.sub.2 CH.sub.2 CH.sub.2                        CH.sub.2 PO.sub.3 H.sub.2        B      35%                        26                                                                                        ##STR54##                       B      60%                        27                                                                                        ##STR55##                       B      50%                        28                                                                                        ##STR56##                       B      65%                        29                                                                                        ##STR57##                       A      20%                        30                                                                                        ##STR58##                       A      80%                        31                                                                                        ##STR59##                       C.sub.1                                                                              80%                        32         A.sub.3 + NH.sub.2 CH.sub.2 CH.sub.2 N(CH.sub.3).sub.2                                                         A      75%                        33                                                                                        ##STR60##                       A      60%                        34                                                                                        ##STR61##                       A.sub.1                                                                              50%                        35         A.sub.3 + NH.sub.3 in MeOH       D      83%                        36         A.sub.3 + NH.sub.2 CH.sub.2 CH.sub.2 CH.sub.2 NH.Boc                                                           A.sub.1                                                                              70%                        37                                                                                        ##STR62##                       A.sub.1                                                                              65%                        38         37 + HCl.NH.sub.2 CH.sub.2 CH.sub.2 COOCH.sub.2 CH.sub.3                                                       Cl     20%                        39         A.sub.3 + TFA.NH.sub.2 CH.sub.2 CH.sub.2 SCH.sub.2 CH.sub.2                   COOCH.sub.3                      A.sub.1                                                                              33%                        40         A.sub.3 + TFA.NH.sub.2 CH.sub.2 CH.sub.2 CH.sub.2 CHCHCOOH                                                     B      51%                        41         A.sub.3 + TFA.NH.sub.2 CH.sub.2 CH.sub.2 OCH.sub.2 CH.sub.2                   COOH                             B      37%                        42         C.sub.2a + HCl.NH.sub.2 CH.sub.2 CH.sub.2 CH.sub.2 CH.sub.2                   CH.sub.2 COOCH.sub.3             F      40%                                   NH.sub.2 CH.sub.2 CH.sub.2 CH.sub.2 CH.sub.2 CH.sub.2 COOH                                                     G      35%                        43         D.sub.1 + HCl.NH.sub.2 CH.sub.2 CH.sub.2 CH.sub.2 CH.sub.2                    CH.sub.2 COOCH.sub.3             H      50%                                   NH.sub.2 CH.sub.2 CH.sub.2 CH.sub.2 CH.sub.2 CH.sub.2 COOH                                                     I      40%                        44         D.sub.2 + HCl.NH.sub.2 CH.sub.2 CH.sub.2 CH.sub.2 CH.sub.2                    CH.sub.2 COOCH.sub.3             J      35%                                   NH.sub.2 CH.sub.2 CH.sub.2 CH.sub.2 CH.sub.2 CH.sub.2 COOH                                                     K      30%                        __________________________________________________________________________     TFA = trifluoroacetic acid                                                    PTSA = ptoluenesulfonic acid                                             

HPLC Analysis

The following table (TABLE III) reports the R_(t) of representativeexamples of compounds of this invention.

Analysis were run with a Varian model 5000 LC pump equipped with a 10 μlloop injector and a Varian 2050 variable wavelength detector at 254 nm.

Columns:

Pre-column LiChroCart-LiChrosorb RP-8 (5 μm) followed by a columnLiChroCart 125-4 LiChrospher 100 RP-8 (5 μm)

Eluents:

A 0.05M aq. HCOONH₄

B CH₃ CN

C THF

Method A:

isochratic 44% of B in A

Flow rate: 0.7 ml/min

Method B:

isochratic 40% of B in A

Flow rate 0.7 ml/min

Method C:

isochratic 38% of B in A

Flow rates 0.5 ml/min

Method D:

isochratic 30% of B in A

Flow rate: 0.7 ml/min

Method E:

isochratic 38% of B in A

Flow rates 0.7 ml/min

Method F:

gradient from 38 to 55% of B in A in 11 min according to the followingprogram

    ______________________________________                                               Time (min)                                                                            % B in A                                                       ______________________________________                                               0       38                                                                    6       38                                                                    7       45                                                                    10      45                                                                    11      55                                                             ______________________________________                                    

Flow rate: 0.7 ml/min

Method G:

gradient from 38 to 55% of B in A in 25 min according to the followingprogram

    ______________________________________                                               Time (min)                                                                            % B in A                                                       ______________________________________                                                0      38                                                                     6      38                                                                    10      44                                                                    15      44                                                                    25      55                                                             ______________________________________                                    

Flow rate: 0.7 ml/min

Method H:

isochratic 55% of B in A

Flow rate: 0.7 ml/min

Method I: isochratic 60% of B in A

Flow rate: 0.7 ml/min

Method L:

isochratic 48% of B in A Flow rate: 0.7 ml/min

Method M:

gradient according to the following program:

    ______________________________________                                        Time (min) % A           % B    % C                                           ______________________________________                                         0         74            10     16                                            20         62            19     19                                            ______________________________________                                    

Flow rate: 0.7 ml/min

                  TABLE III                                                       ______________________________________                                        HPLC Analysis                                                                 Compound No.                                                                            Method   R.sub.t (min)                                                                              K                                             ______________________________________                                         1        A        2.56         0.92                                           2        B        4.09         1.15                                           3        C        6.21         1.12                                           4        D        14.70        1.79                                           5        E        5.99         1.28                                           6        A        4.05         1.46                                           7        A        4.52         1.63                                           8        A        3.44         1.24                                           9        E        5.32         1.18                                          10        E        4.22         0.90                                          11        F        14.30        3.05                                          12        F        5.92         1.28                                          13        E        4.99         1.04                                          14        G        14.09        3.01                                          15        G        17.60        3.76                                          16        G        13.77        2.94                                          17        G        23.75        5.07                                          18        G        7.49         1.60                                          19        F        8.84         1.89                                          20        G        17.77        3.78                                          21        G        31.10        6.64                                          22        F        5.01         1.07                                          23        F        4.40         0.94                                          24        F        6.27         1.34                                          25        F        11.17        2.39                                          26        F        29.04        6.02                                          27        F        6.28         1.34                                          28        F        12.14        2.59                                          29        F        8.02         1.71                                          30        E        6.81-7.61    1.45-1.62                                                        anomeric mixture                                                                           anomeric mixture                              31        F        17.64        3.76                                          32        B        9.64         2.70                                          33        H        15.10        7.40                                          34        I        7.22         3.92                                          35        L        10.28        4.11                                          36        F        19.32        4.13                                          37        F        14.56        3.11                                          38        F        11.00        2.35                                          39        F        9.48         2.02                                          40        F        6.84         1.46                                          41        F        3.95         0.84                                          42        M        17.23         1.32*                                        43        M        15.76         1.52**                                       44        M        16.64          1.50***                                     19        M        20.81        1.32                                          ______________________________________                                         K = Relative Retention time                                                   K = Relative Retention time = R.sub.t amide/R.sub.t GE 2270 proper            starting material (i.e. the compound of formula II wherein W is COOH)    

EXPERIMENTAL SECTION

TABLE IV--N.M.R.

The ¹ H-NMR spectra were recorded with a Bruker spectrometer in DMSO-d₆(hexadeuterodimethylsulfoxide) using TMS as the internal standard (0.00ppm) [δ, ppm, m) at 250 MHz and/or 500 MHz (s=singlet, br s=broadsinglet, d=doublet, dd=doublet of doublets, t=triplet, m=multiplet)

TABLE V--I.R.

The infrared spectra (IR) were recorded with a Perkin Elmer mod. 580spectrophotometer in nujol mull.

TABLE VI--U.V.

The ultraviolet absorption spectra were recorded with a Perkin ElmerModel 320 spectrometer.

It will be clear to the skilled technician that the data represented inTABLES IV, V and VI below, do not represent all the values of the peaksobtained but only the values of those peaks which permit to characterizethe single substance.

                  TABLE IV                                                        ______________________________________                                        N.M.R. Spectra                                                                COMPOUND                                                                      NO.       .sup.1 H--NMR (DMSOd.sub.6) δ(ppm)                            ______________________________________                                         1        0.84(d, 3H); 0.87(d, 3H); 2.57(s, 3H); 3.39(s, 3H);                           3.77(dd, 1H); 3.99(d, 2H); 4.25(dd, 1H); 4.96(s,                              2H); 7.36-7.22(m, 7H); 8.28(s, 1H); 8.50(s, 1H);                              8.59(s, 1H)                                                          2        0.79(d, 3H); 0.85(d, 3H); 2.05-1.70(m, 4H); 2.54(s,                           3H); 3.33(s, 3H); 3.65(m, 2H); 3.81(dd, 1H); 4.10                             (m, 1H), 4.35(dd, 1H); 4.99(s, 2H); 7.35-7.05(m,                              7H); 8.20(s, 1H); 8.42(s, 1H); 8.58(s, 1H)                           3        0.84(d, 3H); 0.87(d, 3H); 2.58(s, 3H); 3.37(s, 3H);                           3.80(dd, 2H); 3,84(dd, 1H); 3.91(dd, 1H); 4.26(dd,                            1H); 4.55(m, 1H); 4.97(s, 2H); 7.36-7.20(m, 7H);                              8.29(s, 1H); 8.55(s, 1H); 8.59(s, 1H)                                4        0.85(d, 3H); 0.89(d, 3H); 2.58(s, 3H); 2.90(m,                                2H); 3.38(s, 3H); 3.70(dd, 1H); 4.29(dd, 1H); 4.85                            (m, 1H); 4.98(s, 2H); 7.40-7.20 (m, 7H); 8.28(s,                              1H); 8.52(s, 1H); 8.58(s, 1H)                                        5        0.85(d, 3H); 0.88(d, 3H); 2.58(s, 3H); 3.11(m, 2H);                           3.26(br; s, 1H); 3.38(s, 3H); 3.78(dd, 1H); 4.28(dd,                          1H)4.64(m, 1H); 4.97(s, 2H); 6.68(d, 1H); 7.09(d,                             1H); 7.40-7.20(m, 7H); 8.27(s, 1H); 8.47(s, 1H);                              8.59(s, 1H)                                                          6        0.84(d, 3H). 0.87(d, 3H); 0.92(d, 3H); 0.95(d, 3H);                           1.69(m, 2H); 1.86(m, 1H); 2.57(s, 3H); 3.37(s,                                3H); 3.78(dd, 1H); 4.26(dd, 1H); 4.53(m, 1H);                                 4.97(s, 2H); 7.38-7.20(m, 7H); 8.28(s, 1H); 8.46(s,                           1H); 8.59(s, 1H)                                                     7        0.84(d, 3H); 0.88(d, 3H); 2.58(s, 3H); 3.20(m,                                2H); 3.37(s, 3H); 3.77(dd, 1H); 4.25(dd, 1H); 4.73                            (m, 1H); 7.40-7.2(m, 12H); 8.28(s, 1H); 8.47(s,                               1H)8.59(s, 1H)                                                       8        0.85(d, 3H); 0.89(d, 3H); 2.08(s, 3H); 2.16(m, 2H);                           2.56(m, 2H); 2.57(s, 3H); 3.40(s, 3H); 3.79(dd,                               1H); 4.27(dd, 1H); 4.61(m, 1H); 5.00(s, 2H); 7.37-                            7.20(m, 7H); 8.29(s, 1H); 8.52(s, 1H); 8.60(s, 1H)                   9        0.84(d, 3H); 0.88(d, 3H); 2.45-1.70(m, 4H); 2.58(s,                           3H); 3.37(s, 3H); 3.68(m, 2H); 3.78(dd, 1H); 4.10                             (m, 1H); 4.27(dd, 1H); 4.49(m, 1H); 7.35-7.22(m,                              7H); 8.27(s, 1H); 8.50(s, 1H); 8.59(s, 1H)                          10        0.85(d, 3H); 0.88(d, 3H); 1.19(d, 3H); 2.59(s, 3H);                           3.39(s, 3H); 3.78(dd, 1H); 4.30(m, 2H); 4.48(dd,                              1H); 4.99(s, 2H); 7.4-7.2(m, 7H); 8.33(s, 1H);                                8.49(s, 1H); 8.60(s, 1H)                                            11        0.84(d, 3H); 0.88(d, 3H): 1.55-1.35(m, 2H); 1.61                              (m, 2H); 1.83(m, 2H); 2.58(s, 3H); 3.34(m, 2H);                               3.38(s, 3H); 3.79(dd, 1H); 3.91(br; s, 1H); 4.29                              (dd, 1H); 4.97(s, 2H); 7.35-7.13(m, 7H); 8.27(s,                              1H); 8.43(s, 1H); 8.59(s, 1H)                                       12        0.85(d, 3H); 0.88(d, 3H); 1.37(m, 2H); 1.70-1.49                              (m, 3H); 1.75(m, 1H); 2.58(s, 3H); 2.76(m, 2H);                               3.38(s, 3H); 3.78(dd, 1H); 4.03(m, 2H); 4.28(m,                               2H); 4.97(s, 2H); 7.35-7.20(m, 7H); 8.28(s, 1H);                              8.49(s, 1H); 8.59(s, 1H)                                            13        0.84(d, 3H); 0.88(d, 3H); 1.98-1.82(m, 2H); 2.18                              (m, 2H); 2.56(s, 3H); 2.69(dd, 2H); 3.36(s, 3H);                              3.85-3.62(m, 3H); 4.31(m, 2H); 4.85(m, 1H); 4.96(s,                           2H); 7.38-7.19(m, 7H); 8.24(s, 1H); 8.55(s, 1H);                              8.63(s, 1H)                                                         14        0.85(d, 3H); 0.88(d, 3H); 1.99-1.82(m, 3H); 2.06                              (m, 1H); 2.58(s, 3H); 3.58(m, 1H); 3.67(m, 1H);                               3.79(dd, 1H); 4.18(d, 2H); 4.28(dd, 1H); 4.97(s,                              2H); 6.93(s, 1H); 7-36-7.28(m, 8H); 8.28(s, 1H);                              8.52(s, 1H); 8.59(s, 1H)                                            15        0.85(d, 3H); 0.88(d, 3H); 2.07-1.63(m, 4H); 2.58(s,                           3H); 2.99(dd, 1H); 3.09(dd, 1H); 3.38(s, 3H); 3.51                            (m, 1H); 3.77(m, 2H); 4.30(m, 2H); 4.89(m, 1H);                               4.98(s, 2H); 6.66(d, 1H); 6.95(br; s, 1H); 7.16(d,                            1H); 7.39-7.20(m, 8H); 8.23(s, 1H); 8.42(s, 1H);                              8.58(s, 1H)                                                         16        0.84(d, 3H); 0.87(d, 3H); 1.20(d, 3H); 1.98-1.80                              (m, 3H); 2.08(m, 1H); 2.56(s, 3H); 3.36(s, 3H);                               3.85-3.71(m, 2H); 4.13(m, 1H); 4.28(dd, 1H); 4.31                             (dd, 1H); 4.73(m, 1H); 5.05(d, 1H); 6.89(br; s,                               1H); 7.15(br; s, 1H); 7.38-7.19(m, 7H); 8.26(s,                               1H); 8.51(s, 1H); 8.56(s, 1H)                                       17        0.84(d, 3H); 0.87(d, 3H); 0.94(d, 3H); 0.98(d, 3H);                           2.10-1.62(m, 7H); 2.56(s, 3H); 3.36(s, 3H); 3.65(m,                           1H); 3.88-3.70(m, 2H); 4.31(m, 2H); 4.88(m, 1H);                              4.96(s, 2H); 6.79(br, s, 1H); 7.18(br; s, 1H); 7.35-                          7.20(m, 7H); 8.25(s, 1H); 8.48(s, 1H); 8.56(s, 1H)                  18        0.84(d, 3H); 0.88(d, 3H); 1.81(m, 2H); 2.30(t, 2H);                           2.58(s, 3H); 3.35(m, 2H); 3.37(s, 3H); 3.78(dd,                               1H); 4.28(dd, 1H); 4.97(s, 2H); 7.35-7.20(m, 7H);                             8.27(s, 1H); 8.46(s, 1H); 8.59(s, 1H)                               19        0.84(d, 3H); 0.87(d, 3H); 1.35(m, 2H); 1.56(m,                                4H); 2.22(t, 2H); 2.58(s, 3H); 3.36(m, 2H); 3.38(s,                           3H); 3.80(dd, 1H); 4.29(dd, 1H); 4.97(s, 2H);                                 7.42-7.22(m, 7H); 8.29(s, 1H); 8.45(s, 1H); 8.62(s,                           1H)                                                                 20        0.84(d, 3H); 0.88(d; 3H); 1.31(br; s, 6H); 1.51(m,                            2H); 1.57(m, 2H); 2.19(t, 2H); 2.58(s, 3H); 3.32                              (m, 2H); 3.37(s, 3H); 3.79(dd, 1H); 4.28(dd, 1H);                             4.97(s, 2H); 7.38-7.19(m, 7H); 8.27(s, 1H); 8.45(s,                           1H); 8.59(s, 1H)                                                    21        0.84(d, 3H); 0.88(d, 3H); 1.41-1.20(m, 12H); 1.47                             (m, 2H); 1.57(m, 2H); 2.17(t, 2H); 2.58(s, 3H);                               3.29(m, 2H); 3.38(s, 3H); 3.79(dd, 1H); 4.28(dd,                              1H); 4.97(s, 2H); 7.38-7.19(m, 7H); 8.27(s, 1H);                              8.43(s, 1H); 8.59(s, 1H)                                            22        0.85(d, 3H); 0.87(d, 3H); 2.57(s, 3H); 2.79(t, 2H);                           3.37(s, 3H); 3.59(t, 2H); 3.78(dd, 1H); 4.28(dd,                              1H); 4.97(s, 1H); 7.41-7.20(m, 7H); 8.27(s, 1H);                              8.44(s, 1H); 8.57(s, 1H)                                            23        0.84(d, 3H); 0.87(d, 3H); 1.67(m, 2H); 2.53(t, 2H);                           2.57(s, 3H); 3.26(t, 2H); 3.37(s, 3H); 3.78(dd, 1H);                          4.28(dd, 1H); 4.97(s, 2H); 7.41-7.26(m, 7H); 8.26                             (s, 1H); 8.44(s, 1H); 8.57(s, 1H)                                   24        0.85(d, 3H); 0.88(d, 3H); 1.58(m, 2H); 1.79(m,                                2H); 2.58(s, 3H); 3.38(s, 3H); 3.50(m, 2H); 3.78                              (dd, 1H); 4.28(dd, 1H); 4.97(s, 2H); 7.38-7.21(m,                             7H); 8.27(s, 1H); 8.45(s, 1H); 8.59(s, 1H)                          25        0.84(d, 3H); 0.88(d, 3H); 1.65-1.35(m, 8H); 4.58(s,                           3H); 3.38(s, 3H); 3.78(dd, 1H); 4.28(dd, 1H); 4.97                            (s, 2H); 7.40-7.20(m, 7H); 8.28(s, 1H); 8.43(s,                               1H); 8.59(s, 1H)                                                    26        0.85(d, 3H); 0.89(d, 3H); 2.56(s, 3H); 3.36(s, 3H);                           3.80(dd, 1H); 4.31(dd, 1H); 4.62(br; s, 2H); 4.96(s,                          2H); 7.39-7.15(m, 7H); 7.47(d, 2H); 7.90(d, 2H);                              8.26(s, 1H); 8.41(s, 1H); 8.58(s, 1H)                               27        0.84(d 3H); 0.88(d, 3H); 1.62(br, s, 2H); 1.92(br,                            s, 2H); 2.58(s, 3H); 2.60(m, 1H); 3.38(s, 3H); 3.79                           (dd, 1H); 4.16(m, 2H); 4.29(dd, 1H); 4.38(m, 2H);                             7.35-7.19(m, 7H); 8.25(s, 1H); 8.29(s, 1H); 8.57(s,                           1H)                                                                 28        0.85(d, 3H); 0.89(d, 3H); 1.39(m, 2H); 1.61(m, 2H)                            1.76(m, 2H); 2,58(s, 3H); 2.88(t, 2H); 3.33(m, 2H);                           3.80(dd, 1H); 4.29(dd, 1H); 4.98(s, 2H); 7.34-7.20                            (m, 7H)8.26(s, 1H); 8.45(s, 1H); 8.58(s, 1H)                        29        0.84(d, 3H); 0.88(d, 3H); 2.58(s, 6H); 3.38(s, 3H);                           3.70-3.41(m, 5H); 3.89-3.75(m, 2H); 3.98(br; s,                               1H); 4.35-4.26(m, 2H); 4.97(s, 2H); 7.35-7.21(m,                              7H); 8.26(s, 1H); 8.28(s, 1H); 8.58(s, 1H)                          30        0.84(d, 3H); 0.88(d, 3H); 2.58(s, 3H); 3.29-3.14                              (m, 2H); 3.38(s, 3H); 3.90-3.49(m, 4H); 4.29(dd,                              1H); 4.92(m, 1H); 4.97(s, 2H); 5.12(t, 1H); 7.35-                             7.18(s, 7H); 8.26(m, 1H); 8.51(s, 1H); 8.58(s, 1H)                  31        0.86(d, 3H); 0.89(d, 3H); 1.81(m, 2H); 2.59(s, 3H);                           3.32(m, 4H); 3.39(s, 3H); 3.80(dd, 1H); 4.30(dd,                              1H); 4.99(s, 2H); 6.75(d, 1H); 7.41-7.18(m, 9H);                              8.28(s, 1H); 8.46(s, 1H); 8.59(s, 1H)                               32        0.85(d, 3H); 0.88(d, 3H); 2.21(s, 6H); 2.59(s, 3H);                           3.38(s, 3H); 3.43(m, 4H); 3.81(dd, 1H); 4.31(dd,                              1H); 4.98(s, 2H); 7.45-7.19(m, 7H); 8.28(s, 1H);                              8.45(s, 1H); 8.61(s, 1H)                                            33        0.86(d, 3H); 0.90(d, 3H); 1.91-1.70(m, 2H); 2.26-                             2.05(m, 2H); 2.60(s, 3H); 2.91-2.69 (m, 4H); 3.40                             (s, 3H); 3.51(br; s, 2H); 3.95-3.75(m, 2H); 4.30                              (dd, 1H); 4.99(s, 2H); 7.41-7.18(m, 12H); 8.28(s,                             1H)8.45(s, 1H); 8.66(s, 1H)                                         34        0.85(d, 3H); 0.89(d, 3H); 1.81-1.49(m, 4H); 2.01-                             1.88(m, 2H); 2.59(s, 3H); 2.98-2.65(m, 4H); 3.39(s,                           3H); 3.80-3.51(m, 4H); 3.81(dd, 1H); 4.31(dd,                                 1H); 4.99(s, 2H); 7.41-7.18(m, 7H); 7.90-7.65(m,                              6H); 8.25(s, 1H); 8.36(s, 1H); 8.61(s, 1H)                          35        0.85(d, 3H); 0.88(d, 3H); 2.59(s, 3H); 3.39(s, 3H);                           3.79(dd, 1H); 4.29(dd, 1H); 4.98(s, 2H); 7.40-7.19                            (m, 7H); 7.72(br, s, 1H); 8.03(br, s, 1H); 8.28(s,                            1H); 8.47(s, 1H); 8.60(s, 1H)                                       36        0.85(d, 3H); 0.88(d, 3H); 1.87(m, 2H); 2.54(s,                                3H); 2.89(m, 2H); 3.37(s, 3H); 3.42(m, 2H); 3.79                              (dd, 1H); 4.29(dd, 1H); 4.98(s, 2H); 7.38-7.20(m,                             7H); 7.69(br; s, 3H); 8.29(s, 1H); 8.49(s, 1H); 8.61                          (s, 1H)                                                             37        0.83(d, 3H); 0.87(d, 3H); 1.32(m, 1H); 2.16(m,                                1H); 2.46(d, 3H); 2.57(s, 3H); 2.71(m, 1H); 3.37(s,                           3H); 3.78(dd, 1H); 4.16(d, 1H); 4.26(dd, 1H); 4.67                            (m, 1H); 4.96(s, 2H); 6.02(d, 1H); 6.35(dd, 1H);                              7.35-7.20(m, 7H); 8.28(s, 1H); 8.49(s, 1H); 8.60(s,                           1H); 9.61(s, 1H)                                                    38        0.83(d, 3H); 0.87(d, 3H); 1.25(m, 1H); 2.2(m, 1H);                            2.5(s, 3H); 2.70(m, 3H); 3.35(s, 3H); 3.63(m, 1H);                            3.79(d, 1H); 4.27(dd, 1H); 4.97(s, 2H); 7.4-7.15                              (m, 7H); 8.28(s, 1H); 8.53(s, 1H); 8.61(s, 1H)                      39        0.83(d, 3H); 0.87(d, 3H); 1.32(m, 1H); 2.16(m,                                1H); 2.47(d, 3H); 2.57(s, 3H); 2.72(m, 4H); 3.37                              (s, 3H); 3.50(m, 2H); 3.78(dd, 1H); 4.27(dd, 1H);                             4.97(s, 2H); 7.40-7.20(m, 7H); 8.28(s, 1H); 8.49(s,                           1H); 8.60(s, 1H)                                                    40        0.83(d, 3H); 0.87(d, 3H); 1.32(m, 1H); 1.71(m,                                2H); 2.25-2.14(m, 1H); 2.46(d, 3H); 2.57(s, 3H);                              2.7(m, 1H); 3.37(s, 3H); 3.76(dd, 1H); 4.27(dd,                               1H); 4.97(s, 2H); 5.81(d, 1H, J=15.7 Hz); 6.78(m,                             1H); 7.39-7.12(m, 7H); 8.28(s, 1H); 8.45(s, 1H);                              8.60(s, 1H)                                                         41        0.86(d, 3H); 0.89(d, 3H); 1.43(m, 1H); 2.19(m, 1H);                           2.47(d, 3H); 2.59(s, 3H); 2.72(m, 1H); 3.39(s, 3H);                           3.50(t, 2H); 3.58(t, 2H); 3.68(t, 2H); 3.79(dd, 1H);                          4.99(s, 2H); 7.42-7.20(m, 7H); 8.27(s, 1H); 8.47                              (s, 1H); 8.59(s, 1H)                                                42        0.83(d, 3H); 0.85(d, 3H); 1.2-1.4(m, 3H); 1.5-1.65                            (m, 4H); 2.22(t, 3H); 2.60(d, 1H); 2.69(d, 1H);                               3.37(s, 3H); 3.79(dd, 1H); 4.27(dd, 1H); 4.86(m,                              2H); 4.97(s, 2H); 5.00(dd, 1H); 5.1-5.4(m, 3H);                               5.74(t, 1H); 6.00(d, 1H); 7.2-7.4(m, 7H); 8.27(s,                             1H); 8.44(s, 1H); 8.62(s, 1H)                                       43        0.84(d, 3H); 0.89(d, 3H); 1.4-1.2(m, 3H); 1.65-1.50                           (m, 4H); 2.23(t, 3H); 2.59(s, 3H); 2.79(m, 1H);                               3.87(m, 1H); 4.25(m, 1H); 5.04(t, 1H); 5.35-5.20                              (m, 3H); 6.09(d, 1H); 6.67(br, s, 1H); 7.04(br, s,                            1H); 7.35-7.15(m, 6H); 8.24(s, 1H); 8.26(s, 1H);                              8.45(s, 1H); 8.61(s, 1H)                                            44        0.84(d, 3H); 0.88(d, 3H); 1.4-1.25(m, 3H); 1.65-                              1.50(m, 4H); 2.23(t, 3H); 2.58(s, 3H); 2.75(m, 1H);                           3.78(dd, 1H): 4.28(dd, 1H); 4.98(m, 3H); 5.35-5.15                            (m, 3H); 6.03(m, 2H); 7.42-7.15(m, 7H): 8.30(s,                               1H): 8.45(s, 1H): 8.62(s, 1H)                                       ______________________________________                                    

                  TABLE V                                                         ______________________________________                                        I.R. Spectra                                                                  COMPOUND NO.                                                                              I.R. (nujol cm.sup.-1)                                            ______________________________________                                         1          3370; 3110; 1730; 1655; 1545; 1520                                 2          3350; 3110; 1720; 1650; 1535; 1500                                 3          3340; 3105; 1720; 1645; 1535; 1500                                 4          3360; 1725; 1640; 1535                                             5          3350; 3110; 1725; 1650; 1535; 1510                                 6          3370; 3105; 1725; 1655; 1535; 1500                                 7          3360; 3100; 1725; 1655; 1535; 1490                                 8          3370; 3105; 1725; 1655: 1535; 1505                                 9          3370; 3110; 3100; 1725; 1657; 1550; 1530; 1505                    10          3370; 3105; 1730; 1655; 1540; 1510                                11          3359; 3115; 1653; 1551; 1510;                                     12          3360; 3113; 1720; 1662; 1547; 1510                                13          3370; 3110; 1720; 1655; 1530; 1505                                14          3350; 3120; 1655; 1535; 1500                                      15          3350; 3100; 1650; 1530; 1510;                                     16          3340; 3105; 1650; 1530                                            17          3340; 3100; 1655; 1530                                            18          3350; 3100; 1710; 1645; 1540                                      19          3360; 3115; 1720; 1665; 1540; 1506;                               20          3350; 3113; 1720; 1659; 1549; 1506                                21          3340; 1710; 1645; 1540; 1500                                      22          3304; 1653; 1540                                                  23          3333; 1657; 1547; 1092; 1038;                                     24          3354; 3113; 1653; 1550; 1506; 1245                                25          3348; 3111; 1660; 1548; 1507; 1245                                26          3315; 1653; 1539; 1238                                            27          3361; 3113; 1720; 1653; 1531; 1507; 1092                          28          3333; 1653; 1547; 1494; 1243                                      29          3356; 3114; 1653; 1508; 1088                                      30          3360; 1670; 1505; 1200                                            31          3351; 3115; 1653; 1549; 1509; 1250                                32          3370; 3110; 1655; 1545; 1500; 1245                                33          3350; 1655; 1530; 1490; 1220                                      34          3360; 3105; 1650; 1545; 1510; 1240                                35          3320; 1747; 1650; 1540; 1225                                      36          3330; 1662; 1547; 1496; 1201                                      37          3327; 1730; 1653; 1464; 1377                                      38          3355; 1720; 1657; 1543; 1377                                      39          3321; 1717; 1652; 1545                                            40          3337; 1665; 1549                                                  41          3341; 1721; 1653; 1548; 1377                                      42          3335; 1722; 1647; 1543                                            43          3317; 1665; 1539                                                  44          3317; 1720; 1649; 1545                                            ______________________________________                                    

                  TABLE VI                                                        ______________________________________                                        U.V. DATA λmax (E.sub.1cm.sup.1%)                                                                   Phosphate                                        Compound                     Buffer                                           No.      MeOH      HCl 0.1N  pH 7.38 KOH 0.1N                                 ______________________________________                                         1       309 (290.9)                                                                             312       309 (247.9)                                                                           309 (252.7)                               2       309 (257.5)                                                                             310 (222.6)                                                                             311     309 (226.3)                               3       309 (297.5)                                                                             312       309 (229.6)                                                                           309 (235.8)                               4       309 (245.1)                                                                             312       308 (234.7)                                                                           308 (234.1)                               5       308 (173.8)                                                                             312       309 (150.0)                                                                           305 (181.2)                               6       309 (277.1)                                                                             313       309 (229.8)                                                                           309 (236.7)                               7       309 (258.6)                                                                             313       309 (207.9)                                                                           309 (218.9)                               8       309 (279.8)                                                                             311       309 (225.9)                                                                           309 (229.4)                               9       309 (261.9)                                                                             313       308 (228.1)                                                                           309 (235.0)                              10       309 (279.3)                                                                             314       309 (241.8)                                                                           309 (251.1)                              11       309 (216.8)                                                                             310 (178.5)                                                                             312     310 (194.9)                              12       309 (226.2)                                                                             310 (188.3)                                                                             311     309 (202.4)                              13       308 (237.9)                                                                             314       308 (247.4)                                                                           308 (260.3)                              14       309 (263.4)                                                                             313       313     314                                      15       309 (222.6)                                                                             313       314     304 (169.8)                              16       309 (235.6)                                                                             313       312     312                                      17       309 (230.3)                                                                             312       312     312                                      18       309 (288.8)                                                                             313       309 (239.4)                                                                           309 (248.0)                              19       309 (283.2)                                                                             312       309 (220.3)                                                                           309 (230.1)                              20       309       314       309     309                                      21       309 (271.6)                                                                             313       311 (221.6)                                                                           309 (221.6)                              22       309 (190.9)                                                                             309 (152.2)                                                                             308 (160.4)                                                                           309 (165.6)                              23       309 (242.2)                                                                             310 (182.2)                                                                             309 (200.9)                                                                           309 (200.9)                              24       309       312       310     309                                      25       309       312       310     309                                      26       309 (260.0)                                                                             313       310 (197.7)                                                                           310 (208.6)                              27       310 (264.6)                                                                             313       310 (227.4)                                                                           310 (232.1)                              28       309 (260.5)                                                                             314       310 (186.8)                                                                           310 (203.6)                              29       309 (243.4)                                                                             311       312     311                                      30       309 (248.5)                                                                             311       311     309                                      31       305 (253.7)                                                                             310       310     313 (249.5)                              32       309 (267.9)                                                                             310 (234.9)                                                                             312     312                                      33       309 (247.5)                                                                             311 (234.4)                                                                             314     312                                      34       310 (224.0)                                                                             309 (198.2)                                                                             310     312                                      35       308 (269.8)                                                                             314       313     313                                      36       309 (243.9)                                                                             309 (205.5)                                                                             312     313                                      37       309 (255.1)                                                                             312       314     312                                      38       308       308       308     308                                      39       308 (247.9)                                                                             312       308 (201.3)                                                                           308 (215)                                40       309 (304.3)                                                                             312       309 (235.9)                                                                           309 (262.0)                              41       309 (256.4)                                                                             312       309 (215.1)                                                                           309 (228.6)                              42       309       312       309     307                                      43       309 (253.6)                                                                             313       309 (208.4)                                                                           309 (235.4)                              44       309 (264.9)                                                                             314       309 (208.1)                                                                           309 (223.7)                              ______________________________________                                    

The antimicrobial activity of the compounds of the invention can bedemonstrated by a series of standard tests in vitro.

MIC for Propionibacterium aches, and Bacteroides fragilis are determinedby agar dilution (inocula 10⁴ /10⁵ CFU/spot). MIC for other organismsare determined by microbroth dilution (inocula 10⁴ to 10⁵ CFU/ml).Incubation times are 18-24 h, except for Haemophilus influenzae, P.acnes, B. fragilis (48 h). All organisms are incubated at 37° C.; H.influenzae is incubated in a 5% CO₂ atmosphere, anaerobes in ananaerobic gas mixture. Media used are: Iso-Sensitest broth (Oxoid)(Staphylococci, Streptococcus faecalis, Escherichia coli, Proteusvulgaris; brain heart infusion broth (Difco)+1% Supplement C (Difco) (H.influenzae);

The minimal inhibitory concentrations (MIC, microgram/ml) for somemicroorganisms are reported below in Table VII.

                                      TABLE VII                                   __________________________________________________________________________    (MIC, microgram/ml)                                                                             COMPOUND OF EXAMPLE No.                                     STRAIN            1    2    6    7    8                                       __________________________________________________________________________    Staph. aureus L165 Tour                                                                         0.5  0.13 <0.13                                                                              0.25 0.25                                    Staph. epidermidis L147 ATCC 12228                                                              1    0.25 1    0.5  2                                       Staph. haemolyticus L602                                                                        4    16   1    4    2                                       Strep. pneumoniae L44 UC41                                                                      8    >128 2    4    4                                       Strep. faecalis L149 ATCC 7080                                                                  0.25 0.06 <0.13                                                                              >0.13                                                                              0.25                                    Prop. acnes L1014 ATCC 6919                                                                     <0.13                                                                              0.06 <0.13                                                                              <0.13                                                                              <0.13                                   Bact. fragilis L1010 ATCC 23745                                                                 8    >128 >128 >128 32                                      Haemophilus Influenzae type B                                                                   8    >128 32   128  64                                      Esch. coli L47 SKF 12140                                                                        >128 >128 >128 >128 >128                                    Prot. vulgaris ATCC 881                                                                         >128 >128 >128 >128 >128                                    __________________________________________________________________________                      COMPOUND OF EXAMPLE No.                                     STRAIN            14   18   19   20   26                                      __________________________________________________________________________    Staph. aureus L165 Tour                                                                         0.06 0.25 0.06 0.25 0.25                                    Staph. epidermidis L147 ATCC 12228                                                              0.13 0.25 0.06 0.25 0.25                                    Staph. haemolyticus L602                                                                        0.25 1    0.25 0.25 0.5                                     Strep. pneumoniae L44 UC41                                                                      >128 1    0.25 2    2                                       Strep. faecalis L149 ATCC 7080                                                                  0.06 0.13 0.06 <0.13                                                                              0.13                                    Prop. acnes L1014 ATCC 6919                                                                     0.03 0.016                                                                              0.008                                                                              0.03 0.008                                   Bact. fragilis L1010 ATCC 23745                                                                 >128 2    4    >128 32                                      Haemophilus Influenzae type B                                                                   >128 2    2    >128 8                                       Esch. coli L47 SKF 12140                                                                        >128 >128 >128 >128 >128                                    Prot. vulgaris ATCC 881                                                                         >128 >128 >128 >128 >128                                    __________________________________________________________________________                       COMPOUND OF EXAMPLE No.                                    STRAIN             27    28    32    35                                       __________________________________________________________________________    Staph. aureus L165 Tour                                                                          0.25  0.13  0.5   0.13                                     Staph. epidermidis L147 ATCC 12228                                                               0.5   0.5   0.5   0.13                                     Staph. haemolyticus L602                                                                         1     1     0.5   0.5                                      Strep. pneumoniae L44 UC41                                                                       8     2     1     >128                                     Strep. faecalis L149 ATCC 7080                                                                   1     0.06  0.25  0.06                                     Prop. acnes L1014 ATCC 6919                                                                      0.03  0.008 0.13  0.004                                    Bact. fragilis L1010 ATCC 23745                                                                  64    >128  >128  >128                                     Haemophilus Influenzae type B                                                                    8     >128  >128  >128                                     Esch. coli L47 SKF 12140                                                                         >128  >128  >128  >128                                     Prot. vulgaris ATCC 881                                                                          >128  >128  >128  >128                                     __________________________________________________________________________                      COMPOUND OF EXAMPLE No.                                     STRAIN            36   37   38   39   40                                      __________________________________________________________________________    Staph. aureus L165 Tour                                                                         0.13 0.03 32   0.13 0.25                                    Staph. epidermidis L147 ATCC 12228                                                              0.13 0.06 32   0.25 0.25                                    Staph. haemolyticus L602                                                                        0.13 0.13 64   2    1                                       Strep. pneumoniae L44 UC41                                                                      4    >128 >128 4    1                                       Strep. faecalis L149 ATCC 7080                                                                  0.13 0.06 16   ≦0.13                                                                       ≦0.13                            Prop. acnes L1014 ATCC 6919                                                                     0.06 0.004                                                                              0.25 ≦0.13                                                                       --                                      Bact. fragilis L1010 ATCC 23745                                                                 >128 >128 >128 8    4                                       Haemophilus Influenzae type B                                                                   >128 >128 >128 1    1                                       Esch. coli L47 SKF 12140                                                                        >128 >128 >128 >128 >128                                    Prot. vulgaris ATCC 881                                                                         >128 >128 >128 >128 >128                                    __________________________________________________________________________                       COMPOUND OF EXAMPLE No.                                    STRAIN             41    42    43    44                                       __________________________________________________________________________    Staph. aureus L165 Tour                                                                          0.25  0,13  0.13  0.13                                     Staph. epidermidis L147 ATCC 12228                                                               0.5   0,13  0.5   0.5                                      Staph. haemolyticus L602                                                                         1     0.5   0.5   1                                        Strep. pneumoniae L44 UC41                                                                       2     1     0.5   1                                        Strep. faecalis L149 ATCC 7080                                                                   ≦0.13                                                                        0.13  0.13  0.06                                     Prop. acnes L1014 ATCC 6919                                                                      ≦0.13                                                                        0.016 0.016 0.016                                    Bact. fragilis L1010 ATCC 23745                                                                  4     >128  >128  >128                                     Haemophilus Influenzae type B                                                                    1     4     >128  >128                                     Esch. coli L47 SKF 12140                                                                         >128  >128  >128  >128                                     Prot. vulgaris ATCC 881                                                                          >128  >128  >128  >128                                     __________________________________________________________________________

In view of their properties, the compounds the invention can be used asactive ingredients in the preparation of medicaments for human or animaltreatment.

In particular, the amide derivatives of the antibiotic GE 2270 compoundsof formula I are antimicrobial agents mainly active against grampositive bacteria and gram positive as well as gram negative anaerobes.

The main therapeutic indication of the antibiotic substances of theinvention is thus in the treatment of infections related to the presenceof microorganisms susceptible to them.

The term "treatment" is intended to encompass also prophylaxis, therapyand cure.

The patient receiving this treatment is any animal in need, includingprimates, in particular humans, and other mammals such as equines,cattle, swine and sheep; and poultry and pets in general.

The compounds of the invention can be administered as such or inadmixture with pharmaceutically acceptable carriers and can also beadministered in conjunction with other antimicrobial agents. Conjunctivetherapy, thus includes sequential, simultaneous and separateadministration of the active compounds in a way that the therapeuticaleffects of the first administered one is not entirely disappeared whenthe subsequent is administered.

A preferred pharmaceutical formulation is represented by a formulationsuitable for a topical application on an intact or damaged skin ormucous membrane. Examples of such formulations are powders, ointments,creams and lotions. The excipients in these formulations are the usualpharmaceutically acceptable vehicles such oleaginous ointment bases(e.g. cetyl esters wax, oleic acid, olive oil, paraffin, spermaceti,starch glycerite); absorbent ointment bases (e.g. anhydrous lanolin,hydrophilic petrolatum), emulsion ointment bases (e.g. cetyl alcohol,glyceryl monostearate, lanolin, stearic acid), water-soluble ointmentbases (e.g. glycol ethers and their derivatives which includepolyethylene glycols, poly(oxy-1,2-ethanediyl)-alpha-hydro-omega-hydroxy-octadecanoate, polysorbates, andpolyethylene glycols mono-stearates).

These formulations may contain other known excipients, such aspreservatives and are prepared as known in the art and reported inreference handbooks such as Remington's Pharmaceutical. Sciences,Seventeenth edition, 1985, Mack Publishing Co.

The compounds of the invention can also be formulated into formulationsuitable for parenteral administration according to procedures known perse in the art. For instance, a compound of the invention formulated withpolypropylene glycol or dimethylacetamide and a surface-active agentsuch as polyoxyethylene sorbitan mono-oleate or polyethoxylated castoroil.

A preferred formulation for parenteral administration includes thefollowing excipients: Cremophor® EL (polyoxyl 35 castor oil USP/NF) 20%,propylene glycol 5-10%.

Preferably, this formulation is used for i.v. administration in thetreatment of any infection involving a microorganism susceptible to anantibiotic of the invention.

An example of a suitable formulation used for I.V. is the following

compound No. 19 100 mg

propylene glycol 1 ml

water for injection q.s. 5 ml

phosphate buffer pH 8-8.5

In the treatment of pseudomembranous colitis or other diseasesattributable to the presence of anaerobes in the gastrointestinal tract,an effective dose of the compounds of the invention may be administeredorally in suitable pharmaceutical form such as a capsule or an aqueoussuspension.

The dosage of the active ingredient depends on many factors whichinclude type, age and conditions of the patient, specific activeingredient and formulation selected for administration, administrationschedule, etc.

In general, effective antimicrobial dosages are employed per single unitdosage form. Repeated applications of these dosage forms, e.g. from 2 to6 times a day, are in general preferred. An effective dosage may be ingeneral in the range 0.5-50 mg/kg body weight/day.

A preferred topic preparation is an ointment containing from 1% to 10%of a compound of the present invention.

Anyway, the prescribing physician will be able to determine the optimaldosage for a given patient in a given situation.

Besides their use as medicaments in human and veterinary therapy, thecompounds of the invention can also be used as animal growth promoters.

For this purpose, a compound of the invention is administered orally ina suitable feed. The exact concentration employed is that which isrequired to provide for the active agent in a growth promotant effectiveamount when normal amounts of feed are consumed.

The addition of the active compound of the invention to animal feed ispreferably accomplished by preparing an appropriate feed premixcontaining the active compound in an effective amount and incorporatingthe premix into the complete ration.

Alternatively, an intermediate concentrate or feed supplement containingthe active ingredient can be blended into the feed. The way in whichsuch feed premixes and complete rations can be prepared and administeredare described in reference books (such as "Applied Animal Nutrition", W.H. Freedman and CO., S. Francisco, USA, 1969 or "Livestock Feeds andFeeding" O and B books, Corvallis, Oreg., USA, 1977).

The following examples further illustrate the invention and should notbe interpreted as limiting it in any way.

EXAMPLES OF THE INVENTION

PROCEDURE A--Reaction of GE 2270 factor A₃ starting material with theselected amine

Example 1:

Preparation of compound no. 15, 29, 30, 32, 33

To a stirred solution of 1 mmol of GE 2270 factor A₃ (prepared asdescribed in European Patent Application Publication No. 406745) in 10ml of dimethylformamide (DMF), 1.2 mmols of the selected amine, 1.4mmols of triethylamine (TEA) and 1.2 mmols of di-phenylphosphorazidate(DPPA) were added at 0° C. (If the salt (chloride, p-toluenesulfonate,etc.) of the selected amine was used, a double amount of TEA had to beused). The temperature was allowed to rise to room temperature andstirring was continued for about 4 h. The reaction mixture was thenacidified with 1N aq HCl to about pH 3 and then diluted with water tocomplete precipitation of the product. The wet solid was dried in airand then purified by flash chromatography on silica gel 60 (230-400 meshASTM--Merck) eluting with 3 to 5% methanol in chloroform. Fractionscontaining the title compound were pooled together and the solventevaporated. Trituration of the solid with ethyl ether yielded the titlecompound as a fine powder.

PROCEDURE A1--Reaction of GE 2270 factor A₃ starting material with theselected amine containing further reactive functional group(s), all ofwhich protected, and subsequent deprotection of the protecting group(s).

Example 2

Preparation of compound no. 34, 36

The reaction was substantially carried out as described in Example 1.Once the reaction product had been purified by flash chromatography, 1mmol of the solid obtained was treated with 7 ml of cold trifluoroaceticacid (TFA). The suspension was swirled for a few minutes until asolution was obtained and TFA was evaporated "in vacuo" in the cold. Thegummy product still containing traces of TFA was then treated with ethylether and the title compound was obtained as the trifluoroacetate saltin the form of a fine powder.

Example 3

Preparation of compound no. 1, 3 to 10, 18 to 21, 39

The reaction was substantially carried out as described in Example 1.Once the reaction product had been purified by flash chromatography, 1mmol of the solid obtained was dissolved in 20 ml of dioxane and 1.2 mlof 1N aq NaOH were added under stirring at room temperature. After 5 hthe solution was acidified with 1N aq HCl to pH 2 and diluted with waterto complete precipitation of the title compound which was filtered offand allowed to dry in air yielding the title compound as a fine powder.

Example 4

Preparation of compound no. 2

The reaction was carried out as described Example 3. Once hydrolysis ofthe ester function had been accomplished and the compound had beenallowed to dry in air, 1 mmols of the solid obtained was dissolved in 20ml of TFA and 50 mmols of thioanisole were added under stirring at roomtemperature as described by Y. Kiso et al., Chem. Pharm. Bull. 28, 673,1980. After 3.5 h, TFA was evaporated "in vacuo" in the cold and theresidue taken up in a minimum amount of 1% methanol in chloroform.Addition of ethyl ether induced the precipitation of the title compoundwhich was filtered, washed with more ethyl ether and dried "in vacuo" toyield the trifluoroacetate salt of the title compound as a fine powder.

Example 4bis

Preparation of compound no. 37

The reaction was substantially carried out as described in Example 1.Once the starting material had disappeared from the reaction mixture,water was added and the precipitate obtained was filtered off, washedwith additional water and allowed to dry in air. The crude material wasthen dissolved in 3 ml of THF and stirred overnight at room temperaturein the presence of 10% aq. HCl. Dilution with water provided completeprecipitation of the product which was filtered off and allowed to dryin air. The solid was then purified by flash chromatography on silicagel 60 (230-400 mesh ASTM--Merck) eluting with 2 to 4% methanol inchloroform. Fractions containing the title compound were pooled togetherand the solvent evaporated yielding pale yellow powder.

PROCEDURE B--Reaction of GE 2270 factor A₃ starting material with theselected amine containing unprotected acid moieties.

Example 5

Preparation of compound no. 19, 22 to 28, 40, 41

1.1 mmol of DPPA were added at 0° C. to a stirred solution of 1 mmol ofGE 2270 factor A₃ and 1.5 mmols of TEA in 10 ml of DMF. The temperaturewas allowed to rise to room temperature and stirring was continued for4.5 more hours. 1.5 Mmols of the selected amine and 2 mmols of TEA werethen added to the solution at room temperature and stirring wascontinued at the same temperature for 5 more hours. (If the selectedamine contained more than one acid function, the amount of TEA wasadjusted so to free the amino group). The reaction mixture was thenacidified with 1N aq HCl to about pH 2 and then diluted with water tocomplete precipitation of the product. The wet solid was dried in airand then purified by flash chromatography on silica gel 60 (230-400 meshASTM--Merck) eluting with 5 to 10% methanol in chloroform. Fractionscontaining the title compound were pooled together and the solventevaporated. Trituration of the solid with ethyl ether yielded the titlecompound as a fine powder.

PROCEDURE B1--Reaction of GE 2270 factor A₃ starting material with theselected amine containing reactive functional group(s), all of which arevariously protected, in addition to the unprotected acid group(s) andsubsequent deprotection of the protecting group(s).

Example 6

Preparation of compound no. 11, 12

The reaction was substantially carried out as described in Example 5.Once the reaction product had been purified by flash chromatography, 1mmol of the solid obtained was dissolved in 20 ml of TFA and 50 mmols ofthioanisole were added under stirring at room temperature. After 3.5 h,TFA was evaporated "in vacuo" in the cold and the residue taken up in aminimum-amount of 1% methanol in chloroform. Addition of ethyl etherinduced the precipitation of the title compound which was filtered,washed with more ethyl ether and dried "in vacuo" to yield thetrifluoroacetate salt of the title compound as a fine powder.

PROCEDURE C--Reaction of selected amide derivatives of GE 2270 factor A₃as starting material with the selected reagent.

Example 7

Preparation of compound no. 14, 15, 16, 17 from compound no. 1, 5, 10, 6respectively

To a stirred solution of 1 mmol of the appropriate amide derivative ofGE 2270 factor A₃ (prepared as described in the previous examples) in 10ml of DMF, 1.2 mmols of the selected amine, 1.4 mmols of TEA and 1.2mmols of DPPA were added at 0° C. (If the salt (chloride,p-toluenesulfonate, etc.) of the selected amine was used, a doubleamount of TEA had to be used). The temperature was allowed to rise toroom temperature and stirring was continued for about 4 h. The reactionmixture was then acidified with 1N aq HCl to about pH 3 and then dilutedwith water to complete precipitation of the product. The wet solid wasdried in air and then purified by flash chromatography on silica gel 60(230-400 mesh ASTM--Merck) eluting with 3 to 5% methanol in chloroform.Fractions containing the title compound were pooled together and thesolvent evaporated. Trituration of the solid with ethyl ether yieldedthe title compound as a fine powder.

PROCEDURE C1--Reaction of the selected amide derivative of the GE 2270factor A₃ as starting material with the selected reagent which containsfurther reactive functional group(s), all of which protected, andsubsequent deprotection of the protecting group(s).

Example 8

Preparation of compound no. 13 from compound no. 3

The reaction was carried out as described in Example 7. Once thereaction product had been purified by flash chromatography, 1 mmol ofthe solid obtained was dissolved in 20 ml of dioxane and 1.2 ml of 1N aqNaOH were added under stirring at room temperature. After 5 h thesolution was acidified with 1N aq HCl to pH 2 and diluted with water tocomplete precipitation of the title compound which was filtered off andallowed to dry in air yielding the title compound as a fine powder.

Example 9

Preparation of compound no. 31 from compound no. 36

To a stirred solution of 1 mmol of the appropriate amide derivative ofGE 2270 factor A₃ (prepared as described in the previous examples) in 10ml of 10% methanolic chloroform, 1.2 mmols of TEA and 1.1 mmols theselected reagent (see table --) were added at room temperature. After 20min the solvent was evaporated "in vacuo" and the residue treated with5% aq Na₂ CO₃. The solid obtained was filtered off, washed with more 5%Na₂ CO₃ and water and finally redissolved in 10 ml of methanol. To thissolution, 0.5 ml of water and 0.1 mmols of p-toluenesulfonic acid wereadded and the reaction mixture was stirred at room temperatureovernight. The solution was then reduced to a small volume (about 2 ml)under vacuum and water was added to precipitate the title compoundwhich, after drying in air, was obtained as a fine powder.

Example 9bis

Preparation of compound no. 38 from compound no. 37

To a stirred solution of 0.23 mmols of the appropriate amide derivativeof GE 2270 factor A₃ (prepared as described in the previous examples) in40 ml of ethanol, 9.2 mmols of acetic acid, 9.2 mmols of sodium acetateand 0.506 mmols of the selected reagent (see table II) were added atroom temperature. After 2 hours 0.46 mmols of NaBH₄ (Fluka) were addedand stirring was continued overnight at the same temperature.Evaporation of the solvent provided a crude material which was washedwith 10 ml of 1N HCl, filtered and allowed to dry in air. The solid wasthen purified by flash chromatography on silica gel 60 (230-400 meshASTM--Merck) eluting with 0 to 10% methanol in dichloromethane. Thefractions containing the methyl ester of the title compound(intermediate) were pooled together and the solvent evaporated providinga solid which was redissolved in 2 ml dioxane and treated overnight witha 1.2 molar excess of 1N NaOH at room temperature. Evaporation of thesolvent gave a solid a which was further purified by trituration with amixture of ethyl acetate:methanol yielding the title compound as a finepowder.

PROCEDURE D--Reaction of GE 2270 factor A₂ starting material with theselected amine

Example 10

Preparation of compound No. 35

1 mmol of GE 2270 factor A₂ (prepared as described in European PatentApplication Publication No. 406745) were dissolved in 10 ml of asaturated solution of methanolic ammonia. The solution was allowed tostand for 3 days at room temperature and then evaporated "in vacuo". Theresidue was taken up in 2 ml of methanol and the title compoundprecipitated with water, filtered off and allowed to dry in air.Trituration with ethyl ether yielded the title compound as a finepowder.

PROCEDURE E--Preparation of a salt of a compound of the invention.

Example 11

Preparation of the arginine salt of compound No. 19

To a suspension of 3 g of compound No. 19 (2.42 mmols) 180 ml ofdioxane, a solution of 423 mg of L-arginine (2.42 mmols) in 120 ml ofwater were added under stirring and the non clear solution was thuslyophilized to recovered the desired salt.

PROCEDURE F--Reaction of GE 2270 component C_(2a) starting material(i.e. the compound of formula II wherein R is methoxymethyl, R₁ ismethyl, R₄ is hydroxymethyl and W is COOH) with the selected amine iscontaining further reactive functional group(s), all of which protected,and subsequent deprotection of the protecting group(s).

Example 12

Preparation of compound no. 42

The reaction was carried out as described in Example 3 using GE 2270component C_(2a) starting material instead of factor A₃.

PROCEDURE G--Reaction of GE 2270 component C_(2a) starting material asdescribed in procedure F with the selected amine containing unprotectedacid moieties.

Example 13

Preparation of compound no. 42

The reaction was carried out as described in Example 5 using GE 2270component C_(2a) starting material instead of factor A₃.

PROCEDURE H--Reaction of GE 2270 component D₁ starting material (i.e.the compound of formula II wherein R and R₁ are hydrogen, R₄ is methyland W is COOH) with the selected amine containing further reactivefunctional group(s), all of which protected, and subsequent deprotectionof the protecting group(s).

Example 14

Preparation of compound no. 43

The reaction was carried out as described in Example 3 using GE 2270component D₁ starting material instead of factor A₃.

PROCEDURE I--Reaction of GE 2270 component D₁ starting material asdescribed in procedure H with the selected amine containing unprotectedacid moieties.

Example 15

Preparation of compound no. 43

The reaction was carried out as described in Example 5 using GE 2270component D1 starting material instead of factor A₃.

PROCEDURE J--Reaction of GE 2270 component D₂ (i.e. the compound offormula II wherein R is hydroxymethyl, R₁ and R₄ are methyl and W isCOOH) starting material with the selected amine containing furtherreactive functional group(s), all of which protected, and subsequentdeprotection of the protecting group(s).

Example 16

Preparation of compound no. 44

The reaction was carried out as described in Example 3 using GE 2270component D₂ starting material instead of factor A₃.

PROCEDURE K--Reaction of GE 2270 component starting material asdescribed in procedure J with the selected amine containing unprotectedacid moieties.

Example 17

Preparation of compound no. 44

The reaction was carried out as described in Example 5 using GE 2270component D₂ starting material instead of factor A₃.

PROCEDURE L--Reaction of a mixture of minor components (C_(2a), D₁, D₂and g) of antibiotic GE 2270 (starting material) with the selected aminecontaining further reactive functional group(s), all of which protected,and subsequent deprotection of the protecting group(s).

Example 18

The reaction was carried out as described in Example 3 using a mixtureof minor components (C_(2a), D₁, D₂ and E) of antibiotic GE 2270starting material instead of factor A₃ and methyl 6-aminocaproatehydrochloride (Fluka). R_(t) (min) refer to HPLC method M reported inthe HPLC analysis section.

When Y═--NH CH₂ CH₂ CH₂ CH₂ CH₂ COOCH₃, R_(t) (min) are respectively43.43 for GE 2270 factor C_(2a), 39.42 for GE 2270 factor D₁, 42.29 forGE 2270 factor D₂ and 37.41 for GE 2270 factor E.

When Y═--NH CH₂ CH₂ CH₂ CH₂ CH₂ COOH, R_(t) (min) are respectively 17.23for GE 2270 factor C_(2a), 15.76 for GE 2270 factor D₁, 16.64 for GE2270 factor D₂ and 15.13 for GE 2270 factor E.

PROCEDURE M--Reaction of a selected mixture of minor components (C_(2a),D₁, D₂ and E) of antibiotic GE 2270 starting material with the selectedamine containing unprotected acid moieties.

Example 19

The reaction was carried out as described in Example 5 using a selectedmixture of minor components (C_(2a), D₁, D₂ and E) of antibiotic GE 2270starting material instead of factor A₃ and 6-aminocaproic acid. (Fluka).

R_(t) (rain) refer to Method M reported in the HPLC analysis section andare respectively 17.23 for GE 2270 factor C_(2a), 15.76 for GE 2270factor D₁, 16.64 for GE 2270 factor D₂ and 15.13 for GE 2270 factor E.

PREPARATION OF THE STARTING MATERIALS

1. The following starting materials have been purchased from Fluka(Fluka, Chemika-Biochemika, Buchs, Switzerland):

Glycine ethyl ester hydrochloride,

L-threonine methyl ester hydrochloride,

L-tyrosine methyl ester hydrochloride,

L-leucine methyl ester hydrochloride,

L-phenylalanine methyl ester hydrochloride,

L-methionine methyl ester hydrochloride,

L-proline methyl ester hydrochloride,

L-threonine methyl ester hydrochloride,

Nα-Cbz-L-lysine,

methyl 4-aminobutyrate hydrochloride,

methyl 6-aminocaproate hydrochloride,

6-aminocaproic acid,

4-(methylamino)benzoic acid,

piperidine-4-carboxylic acid,

N-methyl-D-glucamine,

D(+)-glucosamine hydrochloride,

2-dimethylaminoethylamine,

amino acetaldehyde dimethylacetal,

β-alanine ethyl ester hydrochloride.

The following starting materials have been purchased from Sigma (Sigma,Biochemicals:

2. Organic Compounds, St. Louis, U.S.A.):

Nδ-Cbz-L-ornithine,

L-aspartic acid dimethyl ester hydrochloride.

3. The following starting materials have been purchased from Aldrich(Aldrich, Catalogo Prodotti di Chimica Fine, Milano, Italy):

L-Prolinamide,

taurine,

3-amino-1-propanesulfonic acid,

3-aminopropylphosphonic acid,

4-amino-1-benzylpiperidine.

Production of antibiotic GE 2270 for preparing antibiotic GE 2270factors A, B₁, B₂, C₁, C₂, C_(2a), D₁, D₂, and E

A culture of Planobispora rosea ATCC 53773 is grown on an oatmeal agarslant for two weeks at 28°-30° C. and then used to inoculate 500 mlflasks containing 100 ml of a seed media of the following composition:

Starch 20 g/l

Polypeptone 5 g/l

Yeast extract 3 g/l

Beef extract 2 g/l

Soybean meal 2 g/l

Calcium carbonate 1 g/l

Distilled water q.s. 100 ml (adjusted to pg 7.0 before sterilization)

The flask is incubated on a rotary shaker (200 rpm) at 28°-30° C. for 92h. The obtained culture is then used to inoculate a jar fermentercontaining 4 liters of the same medium and the culture is incubated at28°-30° C. for 48 hours with stirring (about 900 rpm) and aeration(about one standard liter of air per volume per minute).

The obtained broth is transferred to a fermenter containing 50 l of thefollowing production medium:

Starch 20 g/l

Peptone 2.5 g/l

Hydrolyzed casein 2.5 g/l

Yeast extract 3 g/l

Beef extract 2 g/l

Soybean meal 2 g/l

Calcium carbonate 1 g/l

Distilled water q.s. (adjusted to pH 7.0 before sterilization) andincubated for about 72 hours at 28°-30° C.

Antibiotic production is monitored by paper disc agar assay using B.subtilis ATCC 6633 grown on minimum Davis medium. The inhibition zonesare evaluated after incubation overnight at 35° C.

4a) Recovery of crude antibiotic GE 2270

The fermentation mass (50 l) obtained above is harvested and submittedto filtration in the presence of a filter aid (Clarcell).

Antibiotic GE 2270 is found mainly in the mycelium, even if a certainamount of it can be recovered also from the filtrates.

The filtrate is adjusted to about pH 7.0 and extracted with ethylacetate (50 1). The organic phase is separated by centrifugation andconcentrated to a small volume under reduced pressure. The obtained oilyresidue is then treated with petroleum ether to precipitate crudeantibiotic GE 2270 that is collected by filtration and dried. 415 mg ofcrude antibiotic GE 2270 complex is obtained.

The mycelium is extracted twice with 20 l of methanol and the pooledextracts are concentrated under reduced pressure to give an aqueousresidue which is extracted twice with ethyl acetate. Crude antibiotic ifGE 2270 (6.06 g) is precipitated by addition of petroleum ether from theconcentrated organic phase.

4b) Isolation of antibiotic GE 2270 factor A

The crude obtained from the mycelium according to the proceduredescribed above (3 g) is dissolved tetrahydrofuran and concentratedunder reduced pressure in the presence of silica gel (230-400 mesh). Theobtained solid residue is collected and applied to a chromatographycolumn containing 300 g of silica gel (230-400 mesh) prepared inmethylene chloride (CH₂ Cl₂). The column is developed first withmethylene chloride (2 l) and then sequentially with 1.5 l mixturesmethylene chloride and methanol in the following ratios 98/2; 96/4,94/6, 92/8, 90/10 and 88/12 (v/v).

Fractions are collected, analyzed by TLC, HPLC or microbiologicallyagainst B. subtilis and pooled according to their antibiotic content.

The pooled fractions containing antibiotic GE 2270 factor A areconcentrated under reduced pressure to give an oily residue which issolubilized with tetrahydrofuran.

From this solution, antibiotic GE 2270 factor A (600 mg) is precipitatedby adding petroleum ether.

4 bis) isolation of mixtures of minor components of a antibiotic GE 2270

A representative mixture particularly enriched in the minor componentsC_(2a), D₁, D₂ and E was established by HPLC comparison with analyticalsamples of each single component.

R_(t) (min) refer to HPLC method M reported in the HPLC analysis sectionand are 20.55 for GE 2270 factor C_(2a), 17.43 for GE 2270 factor D₁,18.17 for GE 2279 factor D₂, and 16.61 for GE 2270 factor E.

Concentration of this fraction under reduced pressure produced an oilyresidue which was redissolved in tetrahydrofuran and precipitated withpetroleum ether as whitish powder.

4c) Separation and isolation of antibiotic GE 2270 factors B₁, B₂, C₁,C₂, D₁, D₂, and E

Antibiotic GE 2270 factors D₁, D₂ and E are separated and purified fromthe above obtained crude mixture by preparative HPLC using a 250×20 mmcolumn packed with Nucleosil® C18 (silica gel functionalized withoctadecylsilane groups) (5 μm) and eluted with mixtures of Phase A: CH₃CN: tetrahydrofuran: 40 mM HCOONH₄ (40:40:20); Phase B: CH₃CN:tetrahydrofuran:40 mM HCOONH₄ (10:10:80). The antibiotic mixture (6mg) was solubilized in 3 ml of Phase B and 1 ml of Phase A and wasinjected into the HPLC column which was eluted flow rate of 14 ml/minwith a 26:74 mixture of Phase A and B. The eluted fractions werecollected according to the UV adsorption profile at 254 nm. Thefractions of subsequent chromatographic runs having homogeneous contentwere pooled and concentrated under reduced pressure to eliminate CH₃ CN.The residual solution showed antibacterial activity againstStaphylococcus aureus Tour L165 by paper disc assay. These solutionswere lyophilized at least three times to remove completely the HCOONH₄buffer residue from the HPLC phases.

The yields were as follows: antibiotic GE 2270 factor E, 11 mg;antibiotic GE 2270 factor D₁, 12 mg; antibiotic GE 2270 factor D₂, 10mg.

4d) Isolation of a purified mixture containing antibiotic GE 2270 factorC_(2a) in mixture with other GE 2270 factors

The preparations of crude GE 2270 factors from 6 repeated fermentationswere pooled and solubilized; into 12 l of CH₂ Cl₂ : methanol (93:7). Theinsoluble material was removed by filtration and the solution,containing the antibiotic complex, was applied to a 13 kg (230-400 mesh)silica gel column equilibrated in CH₂ Cl₂ :methanol (93:7). AntibioticGE 2270 factor C_(2a) was eluted from the column by eluting with CH₂ Cl₂: methanol (93:7). The fractions containing the antibiotic of theinvention (HPLC analysis) were pooled, were concentrated under reducedpressure and were dried to yield 23.5 g of antibiotic GE 2270 factorC_(2a) in mixture with other minor factors.

A portion (5.5 g) of this preparation was again purified by flashchromatography on a column containing 400 g of silica gel (230-400 mesh)equilibrated in methylene chloride (CH₂ Cl₂). The column was developedfirst with methylene chloride (1 liter) and then sequentially with aseries of mixtures of methylene chloride/methanol in the followingratios (v/v): 96/4 (3 liters); 94/6 (1 liter); 92/8 (2 liters); 90/10 (6liters) and 88/12 (4 liters).

The fractions containing mainly GE 2270 factor C_(2a) (HPLC analysis)were pooled and were concentrated The antibiotic preparation (646 mg)was precipitated upon addition of petroleum ether.

4e ) Isolation of pure antibiotic GE 2270 factor C_(2a)

The purified mixture containing mainly antibiotic GE 2270 factor C_(2a)was further purified by preparative HPLC from the above describedpreparation.

A portion of the above described preparation for the antibiotic (10 mg)was solubilized in 1 ml of Phase A (CH₃ CN: tetrahydrofuran: 40 mMHCOONH₄ --40:40:20) and 1 ml of Phase B (CH₃ CN: tetrahydrofuran: 40 mMHCOONH₄ --10:10:80) and was injected into a HPLC 250×20 mm Hibar column(E. Merck; Darmstadt F. R. Germany) pecked with 7 μm Nucleosil °C18(silica gel functionalized with octadecylsilane groups) which wasequilibrated with a mixture of 40% Phase A and 60% Phase B. The columnwas eluted at 15 ml/min flow rate with 22 minutes linear gradient from40% to 50% of Phase A. The UV detection was 254 nm. The fractions of 10subsequent chromatographic runs containing the pure antibiotic of theinvention were pooled and were concentrated under reduced pressure toeliminate CH₃ CN. Antibiotic GE 2270 factor C_(2a) precipitated fromwater. The precipitate was collected by centrifugation, was washed twicewith distilled water and was dried under vacuum yielding 66 mg of thepure antibiotic.

Preparation of antibiotic GE 2270 factor A₂

Antibiotic GE 2270 factor A (prepared as described above) (86 mg) isdissolved in 17 ml of 95% ethanol and 1.7 ml of acetic acid. Afterincubation at 60° C. for 24 h, the resulting solution is diluted with0.1M sodium phosphate buffer pH 7.5 (100 ml) and adjusted to pH 7.5 with1M sodium hydroxide. Ethanol is removed by evaporation under reducedpressure and the aqueous residue is extracted twice with ethyl acetate(100 ml). The organic phase is concentrated under reduced pressure toobtain a solid residue which is solubilized with tetrahydrofuran andthen precipitated by adding petroleum ether. Antibiotic GE 2270 factorA₂ (62 mg) is obtained with minor amounts of antibiotic GE 2270 factorsA and A₁. Pure antibiotic GE 2270 factor A₂ is obtained by preparativeHPLC as follows:

10 Mg of the above crude product is solubilized in tetrahydrofuran,diluted to the solubility limit With water and then injected into a HPLCsystem with a column (250×20 mm) packed with Nucleosil® C18 (5micrometer) reverse phase silica gel by Stacroma®, eluting with a lineargradient from 64% to 93% of phase B in phase A, in 20 min, at a flowrate of about 15 ml/min. In this system, phase A is a 90:10 (v/v)mixture of 18 mM sodium phosphate pH 7.2 and acetonitrile, while phase Bis a 40:60 (v/v) mixture of 18 mM sodium phosphate pH 7.2 andacetonitrile. Fractions of five consecutive runs are collected and UVmonitored at 330 nm. Fractions which contain substantial amounts ofantibiotic GE 2270 factor A₂, which correspond to the major peaks of theUV elution profile, are pooled and concentrated under reduced pressureto an aqueous phase which is extracted twice with ethyl acetate. Thisorganic layer is then washed with distilled water to remove the residualinorganic salts and concentrated to precipitate a Solid residue that isthen dissolved in tetrahydrofuran and re-precipitated with petroleumether, to obtain pure antibiotic GE 2270 factor A₂ (45 mg).

In European Patent Application Publication No. 406745 are describedother alternative method preparing antibiotic GE 2270 factor A₂ as mainreaction product of antibiotic GE 2270 factor A.

6. Preparation of antibiotic GE 2270 factor A₃

Antibiotic GE 2270 factor A₂ is incubated for 1 h at room temperature in0.5M sodium carbonate. The reaction mixture is then diluted with coldwater and brought to pH 6.5 with hydrochloric acid. The neutralizedsolution contains antibiotic GE 2270 factor A₃ as the main reactionproduct. This antibiotic is extracted from the aqueous phase with ethylacetate and then is precipitated from the concentrated organic phase byadding petroleum ether.

Pure antibiotic GE 2270 factor A₃ is obtained by column chromatographyas described below:

1.5 Grams of crude GE 2270 A₃ is dissolved in 60 ml of a 1/1 (v/v)mixture of methanol and dichloromethane and adsorbed on silica gel(75-230 mesh) by evaporation of the solvents under reduced pressure. Thesolid residue is then put on the top of a silica gel (75-230 mesh)column (bed height 40 cm) equilibrated with dichloromethane. The columnis then eluted with mixtures of methanol in dichloromethane in theorder: 1) 2% methanol (450 ml); 2) 5% methanol (500 ml); 3) 10% methanol(600 ml); 4) 15% methanol (500 ml); 5) 20% methanol (500 ml); 6) 30%methanol (250 ml).

Fractions are collected and monitored by TLC and a microbiological assayon B. subtilis ATCC 6633. Antibiotic GE 2270 factor A₃ is normallypresent in the eluates which contain about 15-20% methanol.

The fractions containing the desired product are pooled and concentratedunder reduced pressure. Upon addition of petroleum ether to the residue,antibiotic GE 2270 factor A₃ precipitates (854 mg of pure product).

7. Preparation of the proper starting material from antibiotic factorsD₁, D₂, E and C_(2a)

By substantially following the same procedure described at points 5 and6 above but starting from the single factors D₁, D₂, E and C_(2a) ofantibiotic GE 2270 instead of factor A, the proper starting materials offormula III wherein W is COOH or an activated ester, R is hydrogen orCH₂ OH, R₁ is CH₃ or hydrogen and R₄ is hydroxymethyl or methyl, areobtained.

7a) Preparation of proper starting material from a mixture of minorcomponents (C_(2a), D₁, D₂ and E) of antibiotic GE 2270

By substantially following the same procedure described at point 5 and 6above but starting from a mixture of minor components (C_(2a), D₁, D₂and E) of antibiotic GE 2270 instead of the single factor A, the properstarting material of formula III wherein W is COOH or an activated esterand R, R₁ and R₄ are respectively methoxymethyl, methyl andhydroxymethyl for C_(2a), hydrogen, hydrogen and methyl for D₁,hydroxymethyl, methyl and methyl for D₂ and hydroxymethyl, hydrogen andmethyl for E are obtained.

R_(t) (min) refer to HPLC method M reported in the HPLC analysissection.

When W is an activated ester, R_(t) (min) are respectively 22.51 for GE2270 factor C_(2a), 19.80 for GE 2270 factor D₁, 20.41 for GE 2270factor D₂ and 18.92 for GE 2270 factor E.

When W is COOH, R_(t) (min) are respectively 12.99 for GE 2270 factorC_(2a), 10.38 for GE 2270 factor D₁, 11.08 for GE 2270 factor D₂ and9.03 for GE 2270 factor E.

8. Preparation of glycyl-Nε-Cbz-L-lysine trifluoroacetate

4.8 ml of DPPA (22 mmols) was added at 0° C. to a well stirred solutionof 3.5 g of BOC-glycine (Fluka): (20 mmols) and 7.28 g of Nε-Cbz-L-lysine methyl ester hydrochloride (Fluka) (22 mmols) in 50 ml ofdry DMF. To this solution, a solution of 5.8 ml of TEA (42 mmols) in 50ml of dry DMF was added at 0° C. over a 10-15 min period. Stirring wascontinued for 2 more hours at 0° C. and then overnight at roomtemperature. The reaction mixture was diluted with 250 ml of toluene and500 ml of ethyl acetate and washed with 1N aq. HCl (×3), water, asaturated solution of NaHCO₃ and brine. Drying over Na₂ SO₄ andevaporation of the solvent yielded 9.7 g of a thick oil which resistedany attempt of crystallization. NMR of this oil was in perfect agreementwith the structure of BOC-glycyl-N εCbz-L-lysine methyl ester.

The oil was dissolved in 200 ml of acetone/dioxane 1:1 and 22 ml of 1Naq. NaOH were added over a 30 min period at 0°C. under stirring. Thereaction was then stirred for 45 min at room temperature, diluted with300 ml of cold water, acidified with 25 ml of 1N aq HCl and extractedwith chloroform (×3) and ethyl acetate (×3). Drying over Na₂ SO₄ andevaporation of the solvent yielded 9.4 g of a gum which resisted anyattempt of crystallization. NMR of this gum was in perfect agreementwith the structure of BOC-glycyl-N ε-Cbz-L-lysine.

The gummy compound was treated with 20 ml of cold trifluoroacetic acid(TFA). The reaction mixture was swirled at room temperature until allthe compound went in solution. The solution was reduced to a smallvolume under vacuum in the cold and then ethyl ether was added to induceprecipitation of the title compound. 9.6 g of glycyl-N ε-Cbz-L-lysinetrifluoroacetate were obtained as a white powder. NMR was in perfectagreement with the structure.

Preparation of L-tyrosyl-L-prolinamide.

0.48 ml of DPPA (2 mmols) were added at 0° C. to a well stirred solutionof 538 mg of BOC-L-tyrosine (Fluka) (2 mmols), 228.3 mg of L-prolinamide(Aldrich) (2 mmols) and 168 mg of NaHCO₃ in 5 ml of dry DIFF. Thereaction was stirred for 24 h at room temperature and then diluted with50 ml of water and extracted with chloroform (×3). The organic phase waswashed water, dried over Na₂ SO₄ and the solvent evaporated to yield anoil which was purified by flash chromatography on silica gel 60 (230-400mesh ASTM-Merck) eluting with hexane/acetone 2:3. 420 mg ofBOC-L-tyrosyl-L-prolinamide were in this way obtained as a white solid.NMR was in agreement with the structure.

The solid obtained was dissolved in 6 ml of ethyl acetate and stirredfor 48 h at room temperature in the presence of 4 ml of 3N aq. HCl Thereaction mixture was then evaporated to dryness in vacuo and the residueredissolved in ethanol was precipitated with ethyl ether. 302 mg ofL-tyrosyl-L-prolinamide were obtained as a white powder. NMR was inperfect agreement with the structure.

10. Preparation of methyl 8-aminocaprylate and methyl11-aminoundecanoate p-toluenesulfonates

A solution of 40 mmols of the selected amino acid (Fluka) and 15.2 g ofp-toluenesulfonic acid monohydrate (Fluka) (80 mmols) in 200 ml ofmethanol was refluxed overnight. The solvent was then evaporated invacuo and the residue redissolved in ethyl ether. After sometime thetitle compounds crystallized out quantitatively. The NMR of bothcompounds was in agreement with their structure.

11. Preparation of 5-aminopentylphosphonic acid

3.48 g of 5-amino-1-pentanol (Fluka) (33.7 mmols) and 5.0 g of phthalicanhydride (Fluka) (33.7 mmols) were melted together at 180° C. Thistemperature was maintained for 90 min until no more water developed. Thereaction was allowed to cool to room temperature and the oily mixturewas chromatographed on silica gel 60 (230-400 mesh ASTM-Merck) elutingwith 2% methanol in chloroform. 5.9 g of a pure oil were obtained. NMRwas in agreement with the structure.

To the 5.9 g of the oily intermediate (25 mmols), 1.6 ml of PBr₃ (17mmols) were added portionwise so to control the exothermic reaction. Thereaction mixture was heated at 100° C. for 1.5 h and then poured intocrushed ice. The solid material that separated was filtered and allowedto dry in air overnight. 6.6 g of the pure bromo intermediate wereobtained. The mass was in agreement with the expected molecular weight.

500 mg of the pure bromo intermediate (1.69 mmols) and 140 mg oftriethyl phosphite (Fluka) (0.84 mmols) were heated together at 150° C.for about 1 h. Other three portions of 140 mg of triethyl phosphite werethen added at 30 min interval at the same temperature. When all thestarting material had disappeared, the excess of triethyl phosphite wasdistilled off and the crude material purified by flash chromatography onsilica gel 60 (230-400 mesh ASTM-Merck) eluting with 2% methanol indichloromethane. 468 mg of the expected diethyl phosphonate wereobtained as a thick oil. NMR confirmed the structure.

468 mg of the diethyl phosphonate intermediate were treated overnightwith 3 ml of a 0.2M solution of hydrazine in methanol at roomtemperature. The precipitated phthalhydrizide was filtered off and theremaining solution was evaporated to dryness in vacuo. The residue wastaken up in 1N aq. HCl and the solution was washed with ethyl acetate,basified with NaOH and extracted several times with n-butanol. Thebutanolic phase was dried over Na₂ SO₄ and evaporated to dryness toyield 175 mg of a thick oil whose NMR was in agreement with the productexpected.

175 mg of diethyl 5-aminopentylphosphonate were refluxed for 20 h in 0.6ml of conc. HCl. The acid solution was then evaporated to dryness byazeotropic distillation in vacuo in the presence of n-butanol. The NMRof the glassy oil obtained confirmed it to be the5-aminopentylphosphonic acid.

12. Preparation of 5-(5-aminopentyl)tetrazole

To a solution of 10 ml of 6-aminocapronitrile (Fluka) (80 mmols) and13.3 ml of TEA (96 mmols) in 80 ml of tetrahydrofuran, 12.48 ml ofbenzyl chloroformate (Fluka) (88 mmols) were added dropwise at 0° C.under stirring. Stirring was continued for 2 h at room temperature andthe solvent was evaporated in vacuo. The residue was dissolved in ethylacetate, washed with 1N aq. HCl, water and then dried over Na₂ SO₄ andthe solvent evaporated to yield 19.6 g of a syrup whose NMR was inagreement with the structure.

1 g of the protected 6-aminocapronitrile (4.06 mmols) in 40 ml of1-methyl-2-pyrrolidone was heated at 150° C. under argon in the presenceof 793 mg of sodium azide (12.2 mmols) and 834 mg triethylaminehydrochloride (6.1 mmols). After 4 h the reaction mixture was dilutedwith 120 ml of water and then carefully acidified to pH 1 with 10% aq.HCl (attention: azotidric acid forms!). The solution was extracted withethyl acetate, the organic phase re-extracted with 10% aq. NaOH (×2) andthe basic solution washed with ethyl ether, acidified with conc. HCl andextracted with ethyl acetate (×3). Drying and evaporation of the organicphase yielded a syrup that crystallized from methanol/water. 260 mg of afine powder were obtained. NMR and mass confirmed the structure.

250 mg of the N-protected amino tetrazole (0.86 mmols) were treated atroom temperature with 5 ml of thioanisole (43.25 mmols) and 17.5 ml oftrifluoroacetic acid for 3 h. Trifluoroacetic acid was concentrated invacuo in the cold and ethyl ether was added to precipitate the titlecompound as its trifluoroacetate salt. NMR and mass confirmed thestructure.

13. Preparation ofN-[3,4-di-(O-tetrahydropyranyl)benzoyl]-thiazolidin-2-thione

A solution of 4.62 g of 3.4-dihydroxybenzoic acid (Fluka) (30 mmols) in40 ml of methanol was refluxed for 24 h in the presence of 0.325 ml ofconc. H₂ SO₄. After cooling the solution to room temperature some solidNaHCO₃ was added and the solvent evaporated in vacuo. The residue wastaken up in ethyl acetate, washed with water, dried over Na₂ SO₄ and thesolvent evaporated to yield a syrup which was crystallized from ethylacetate/hexane. 3.53 g of white crystals were obtained.

9.1 ml of dihydropyrane (Fluka) (0.1 mol) and 250 mg of pyridiniump-toluenesulfonate (1 mmol) were added at room temperature to a stirredsolution of 1.68 g of methyl 3,4-dihydroxybenzoate (10 mmols) in 4 ml ofethyl acetate and 25 ml of dichloromethane. After 4 d the reactionmixture was washed with a saturated solution of NaHCO₃, dried over Na₂SO₄ and evaporated to dryness to obtain 3.36 g of an oil which was usedfor the next step without further purification.

The crude from the previous reaction was dissolved in 40 ml of acetoneand to the stirred solution 20 ml of water, 2.76 g of K₂ CO₃ (20 mmols)and 10 ml of 1N aq. NaOH (10 mmols) were added and stirring wascontinued for 7 d at room temperature. Acetone was evaporated in vacuoand the residual water phase was washed with ethyl acetate. The aqueousphase was transferred to an E. flask containing an equal volume ofchloroform, cooled to 0° C. and carefully acidified under vigorousstirring with 50 ml of 1N aq. HCl. The water phase was then extracted 3more times with chloroform and the combined organic layers were washedwith 0.2% ammonium formate, dried over Na₂ SO₄ and evaporated to drynessto yield a syrup which crystallized after hexane addition. 2.34 9 of awhite solid were obtained. The NMR was in agreement with the structure.

333 mg of 2-thiazoline-2-thiol (Fluka) (2.8 mmols), 577 mg ofN,N'-dicyclohexylcarbodiimide (Fluka) (2.8 mmols) and 35 mg of4-dimethylamino-pyridine were added in the order at 0° C. to a stirredsolution of 644 mg of the benzoic acid intermediate (2 mmols) in 14 mlethyl acetate/dichloromethane 5:2. Stirring was continued overnight atroom temperature, the precipitated dicyclohexylurea was filtered off andthe yellow solution was evaporated in vacuo to yield yellow oil whichwas purified by flash chromatography on silica gel 60 (230-400 meshASTM-Merck) eluting from 25% acetone in hexane. 708 mg of yellowcrystals were obtained from acetone/hexane. NMR and IR confirmed thecompound to be the title compound.

14. Preparation of N¹ -N⁸ -di-tert-butoxycarbonylspermidine

A solution of 19.72 g of BOC-ON (Aldrich) (80 mmols) in 60 ml ofdegassed tetrahydrofuran (THF) was added dropwise over a 1 h periodunder argon to a stirred solution of 5.8 g of spermidine (Aldrich) (40mmols) in 40 ml of degassed THF cooled at 0° C. The reaction was thenstirred at room temperature overnight and then evaporated to dryness.The residue was taken up in ethyl ether, washed with 1N aq. NaOH (×4)and water (×4), dried over Na₂ SO₄ and the solvent concentrated to asmall volume in vacuo. Upon addition of ethyl ether 11 g of a whitepowder precipitated. NMR confirmed it to be the title compound.

15. Preparation of N-tert-butoxycarbonylpropilendiamine

4.2 g of BOC-ON (Aldrich) (17.2 mmols) were added at room temperature toa stirred solution of 2 g of 3-aminopropionitrile fumarate (Aldrich)(15.6 mmols) dissolved in a mixture of 10 ml of dioxane, 10 ml of waterand 3.3 ml of triethylamine. After 3 h the reaction mixture was dilutedwith more water and extracted with dichloromethane (×3). The combinedorganic layers were washed with 1N aq. NaOH (×3) and water (×3), driedover Na₂ SO₄ and evaporated to dryness. The residual oil was taken up inethyl ether and precipitated with hexane to yield 2.2 g of a whitepowder.

1 g of N-BOC-protected intermediate (5.9 mmols) in 7 ml of 1N ethanolicNaOH was hydrogenated at 40 psi in the presence of 130 mg of Raneynickel (50% slurry in water, pH>9) (Aldrich) for 40 h. Raney nickel wasfiltered off and the solvent was evaporated to dryness. The residue wastaken up in ethyl acetate and washed with 1 N aq. NaOH, dried over Na₂SO₄ and the solvent removed in vacuo yielding 950 mg of a colorless oilwhich solidified on standing. NMR confirmed it to be the title compound.

16. Preparation of 3-(2-aminoethylthio)propanoic acid methyl estertrifluoroacetate

To a solution of 0.5 g of cysteamine (Fluka) (6.48 mmols) in 5 ml of CH₂Cl₂, 1.4 g of di-tert-butyl dicarbonate (Aldrich) (6.48 mmols) in 5 mlof CH₂ Cl₂ were added at room temperature under stirring. After 30 minthe organic solvent was evaporated and the crude material dissolved in 5ml of absolute ethanol. To the ethanolic solution, 2.7 ml of TEA (19.1mmols) and 1.07 ml of methyl 3-bromopropionate (Fluka) (9.57 mmols) wereadded in the order. The reaction was completed in about 30 min. Ethanolwas removed in vacuo and replaced by 15 ml of chloroform. The organicphase was then washed with water, anidrified on Na₂ SO₄ and solventevaporated to yield an oil which was finally treated with 1 ml oftrifluoroacetic acid at 0° C. for 5 min. Evaporation to dryness gave 270mg of a pale yellow oil. NMR and IR confirmed it to be the titlecompound

17. Preparation of 6-amino-2(E)-hexenoic acid

To a stirred solution of 2 ml of 4-amino-butyraldehyde diethyl acetal(Fluka) (11.6 mmols) and 3.6 ml of TEA (25.6 mmols) in 5 ml of CH₂ Cl₂,a solution of 1.5 ml of benzoyl chloride (Fluka) (12.9 mmols) in 5 mlCH₂ Cl₂, was added in 30 min. at room temperature. After 1 hour thereaction was diluted with 10 more ml of CH₂ Cl₂, washed with water andthe organic phase dried over Na₂ SO₄ and the volume adjusted to 20 ml.The new solution was allowed to react for three days under argon in thepresence of 1.6 ml of TEA (11.5 mmols), 10.2 g of di-tert-butyldicarbonate (Aldrich) (46.8 mmols) and 1.4 g of 4-dimethylamino-pyridine(Fluka) (11.5 mmols) at room temperature. Removal of the solvent gave abrown oil that was purified by flash chromatography on silica gel 60(230-400 mesh ASTM-Merck) eluting with 20% ethyl acetate in n-hexaneyielding 1.6 g of the N,N deprotected 4-aminobutyraldehyde diethylacetal as a colorless oil. NMR confirmed the structure.

The obtained oil was then dissolved in 5 ml of THF and treated with 5 mlof 1N HCl at room temperature for three hours. THF was removed in vacuoand the remaining solution was washed with chloroform (2 ml×3). Theorganic phase was then washed with a solution of Na₂ CO₃, water, driedover Na₂ SO₄ and evaporated to dryness yielding an oil that was used inthe next step without further purification.

To a suspension of 160 mg of 60% NaH (4 mmols) in 5 ml of dry THF at 0°C. under argon, 0.837 ml of triethylphosphonoacetate (Fluka) (4.3 mmols)were added. After 30 min. a dry THF (2 ml) solution of the previouslyobtained aldehyde (1.17 g) (4.02 mmols) was added and the temperaturewas allowed to rise to room temperature. The reaction was stirredovernight and then 50 more mg of 60% NaH were added at 0 C. After twomore hours at room temperature the reaction mixture was treated withdiluted HCl (10 ml) and extracted with ethyl acetate (5 ml×3). Thecombined organic phase was washed with water, dried over Na₂ SO₄ andevaporated to dryness. The crude material was purified by flashchromatography on silica gel 60 (230-400 mesh ASTM-Merck) eluting with15% ethyl acetate in n-hexane yielding 765 mg of a syrup. NMR confirmedit to be the expected product with the double bond in E configuration(J=16 Hz).

6.15 ml of 1N LiOH (6.15 mmols) were added to a solution of 739 mg ofthe unsaturated ester previously obtained (2.05 mmols) in 10 ml of THFunder stirring at room temperature. When the starting material haddisappeared the reaction mixture was concentrated in vacuo at 30° C.(bath temperature). The aqueous solution was acidified at pH 2 with 1NHCl and then extracted with ethyl acetate. The combined organic phasewas dried over Na₂ SO₄, filtered and the solvent evaporated yielding anoil that solidified upon standing under vacuum. NMR and MS confirmed itto be 6-N-BOC-amino-2(E)-hexenoic acid.

Removal of the N-BOC protection to obtain the title compound was carriedout in neat trifluoroacetic acid at 0°C. just before the coupling withthe appropriate GE 2270 starting material.

18. Preparation of 3-(2-aminoethoxy)propanoic acid trifluoroacetate

To a stirred solution of 1 g of N-BOC-ethanolamine (6.22 mmols)[prepared according to classical methodologies from ethanolamine(Fluka)] in 10 ml of dry THF at -78 C., 3.88 ml of 1.6M solution ofbutyllithium (Fluka) (6.22 mmols) were added under argon. After 30 min.1.3 g of t-butyl 3-bromo propanoate [prepared according to classicalmethodologies from 3-bromo propanoic acid (Fluka)] (6.22 mmols) wereadded, the temperature allowed to rise to room temperature and theresulting mixture stirred for 20 hours at that temperature. Afterdilution with water the reaction mixture was extracted with n-hexane (5ml×2). Removal of the solvent gave a crude material that was purified byflash chromatography on silica gel 60 (230-400 mesh ASTM-Merck) elutingwith 20% ethyl acetate in n-hexane yielding 1.43 g of an oil. NMRconfirmed it to be the coupled compound.

The total deprotection of the coupled compound was carried outimmediately before addition to the appropriate GE 2270 starting materialby stirring it in trifluoroacetic acid for about 5 min at roomtemperature. Removal of trifluoroacetic acid in vacuo yielded the titlecompound.

We claim:
 1. An amide derivative of antibiotic GE 2270 having the following formula I ##STR63## wherein R represents:hydrogen, hydroxymethyl, or methoxymethyl; R₁ represents:hydrogen, or methyl; Y represents:a group of formula ##STR64## wherein: R₂ represents: hydrogen, (C₁ -C₄)alkyl, amino(C₂ -C₄)alkyl, (C₁ -C₄)alkylamino-(C₁ -C₄)alkyl, or di-(C₁ -C₄)alkylamino-(C₁ -C₄)alkyl; R₃ represents:hydrogen; a linear or branched (C₁ -C₁₄)alkyl group bearing from 1 to 3 substituents selected from: carboxy, sulfo, phosphino, amino which may be optionally protected with a lower alkoxycarbonyl or a benzyloxycarbonyl group, (C₁ -C₄)alkylamino wherein the alkyl moiety may be optionally substituted with a carboxy group, di-(C₁ -C₄)alkylamino, hydroxy, halo, (C₁ -C₄)alkoxy wherein the alkyl moiety may be optionally substituted with a carboxy group, (C₁ -C₄)alkoxycarbonyl, mercapto, (C₁ -C₄)alkylthio wherein the alkyl moiety may be optionally substituted with a carboxy group, phenyl which may be optionally substituted with 1 to 3 substituents selected from carboxy, sulfo, hydroxy, halo and mercapto, carbamyl, (C₁ -C₆)alkylcarbamyl wherein the alkyl moiety may be optionally substituted with 1 or 2 substituents selected from carboxy, amino, (C₁ -C₄)alkylamino and di-(C₁ -C₄)alkylamino, di-(C₁ -C₄)alkylcarbamyl wherein the alkyl moieties together with the adjacent nitrogen atom may also represent a saturated 5-7 membered heterocyclic ring which may optionally be substituted with a carboxy or a carbamyl group on one of the ring carbons and may optionally contain a further heterogroup selected from O, S and N, benzoylamino wherein the phenyl group may be substituted from 1 to 3 hydroxy group, a nitrogen containing 5-6 membered heterocyclic ring which may unsaturated, partially saturated or wholly saturated and may contain 1 to 3 further heteroatoms selected from N, S and O wherein one of the carbons of the ring may optionally bear a group carboxy, sulfo carboxy(C₁ -C₄)alkyl or sulfo(C₁ -C₄)alkyl and the ring nitrogen atom may optionally be substituted by (C₁ -C₄)alkyl, carboxy(C₁ -C₄)alkyl, sulfo(C₁ -C₄)alkyl, or benzyl; (C₃ -C₆)alkenyl, optionally substituted by carboxy or sulfo; 1-deoxy-1-glucityl; 2-deoxy-2-glucosyl; a fully saturated 5 to 7 membered nitrogen containing heterocyclic ring wherein the nitrogen atom may be optionally substituted by (C₁ -C₄)alkyl or benzyl and one or two carbons of the ring skeleton may bear a substituent selected from (C₁ -C₄)alkyl, carboxy and sulfo;or R₂ and R₃ taken together with the adjacent nitrogen atom represent a fully saturated 5-7 membered heterocyclic ring which may optionally contain a further heteroatom selected from O, S and N, and may optionally bear one or two substituents on the ring carbons selected from (C₁ -C₄)alkyl, benzyl, carboxy, sulfo, carboxy(C₁ -C₄)alkyl, and sulfo(C₁ -C₄)alkyl; R₄ represents:hydrogen, methyl, or hydroxymethyl;with the proviso that when R₄ is hydrogen or hydroxymethyl, then simultaneously R is methoxymethyl and R₁ is methyl; and the pharmaceutically acceptable addition salts thereof.
 2. A compound according to claim 1 wherein R represents methoxymethyl and the other substituents are defined as in claim
 1. 3. A compound as claimed in claim 1 wherein R represents methoxymethyl, R₁ and R₄ represent methyl and Y represents a group of formula ##STR65## wherein R₂ is hydrogen and R₃ is defined as in claim
 1. 4. A compound as claimed in claim 1 wherein R is methoxymethyl, R₁ and R₄ represent a methyl group and Y is an amino moiety which is derived from a natural amino acid selected from the group consisting of glycine, ornithine, serine, aspartic acid, tyrosine, leucine, phenylalanine, methionine, proline, threonine, and lysine, or a synthetic dipeptide selected from the group consisting of glycyllysine, serylproline, glycylprolinamide, tyrosylprolinamide, threonylprolinamide, and leucylprolinamide.
 5. A compound as claimed in claim 1 wherein R is methoxymethyl, R₁ and R₄ are methyl, Y is a group NR₂ R₃ wherein R₂ is hydrogen and R₃ is a linear alkyl chain of 3 to 12 carbons substituted with a group selected from COOH, SO₃ H and PO₃ H₂.
 6. A compound as claimed in claim 1 wherein R is methoxymethyl, R₁ and R₄ are methyl, Y is a group NR₂ R₃ wherein R₂ is hydrogen and R₃ is CH₂ CH₂ CH₂ CH₂ CH₂ --COOH.
 7. A compound as claimed in claim 1 wherein R represents hydrogen, hydroxymethyl or methoxymethyl, R₁ represents hydrogen or methyl, R₄ represents hydrogen, methyl or hydroxymethyl and Y represents a group of formula ##STR66## wherein R₂ is hydrogen and R₃ is defined as in claim
 1. 8. A compound as claimed in claim 7 wherein Y is an amino moiety which is derived from a natural amino acid selected from the group consisting of glycine, ornithine, serine, aspartic acid, tyrosine, leucine, phenylalanine, methionine, proline, threonine, and lysine, or a synthetic dipeptide selected from the group consisting of glycyllysine, serylproline, glycylprolinamide, tyrosylprolinamide, threonylprolinamide, and leucylprolinamide.
 9. A compound as claimed in claim 1 wherein R is hydrogen, hydroxymethyl or methoxymethyl, R₁ is hydrogen or methyl, R₄ is hydrogen, methyl or hydroxymethyl and Y is a group NR₂ R₃ wherein R₂ is hydrogen and R₃ is a linear alkyl chain of 3 to 12 carbons substituted with a group selected from COOH, SO₃ H and PO₃ H₂.
 10. A compound as claimed in claim 1 wherein R is hydrogen, hydroxymethyl or methoxymethyl, R₁ is hydrogen or methyl, R₄ is hydrogen, methyl or hydroxymethyl and Y is a group NR₂ R₃ wherein R₂ is hydrogen and R₃ is CH₂ CH₂ CH₂ CH₂ CH₂ --COOH.
 11. A compound according to claim 5 in which R₃ is represented by a linear alkyl chain of 3 to 7 carbons.
 12. A compound according to claim 9 in which R₃ is represented by a linear alkyl chain of 3 to 7 carbons.
 13. A pharmaceutical composition containing a compound of claim 1 as the active ingredient in admixture with a pharmaceutically acceptable carrier. 